U.S. patent application number 09/135238 was filed with the patent office on 2002-11-28 for toso.
Invention is credited to HITOSHI, YASUMICHI, NOLAN, GARRY P..
Application Number | 20020177565 09/135238 |
Document ID | / |
Family ID | 26746328 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177565 |
Kind Code |
A1 |
NOLAN, GARRY P. ; et
al. |
November 28, 2002 |
TOSO
Abstract
The present invention is directed to novel polypeptides such as
the the Toso protein and related molecuels which have an inhibitory
effect on TNF mediated apoptosis and to nucleic acid molecules
encoding those polypeptides. Also provided herein are vectors and
host cells comprising those nucleic acid sequences, chimeric
polypeptide molecules comprising the polypeptides of the present
invention fused to heterologouspolypeptide sequences, antibodies
which bind to the polypeptides of the present invention and to
methods for producing the polypeptides of the present
invention.
Inventors: |
NOLAN, GARRY P.; (PALO ALTO,
CA) ; HITOSHI, YASUMICHI; (MOUNTAIN VIEW,
CA) |
Correspondence
Address: |
FLEHR HOHBACH TEST
ALBRITTON & HERBERT
4 EMBARCADERO CENTER SUITE 3400
SAN FRANCISCO
CA
94111
|
Family ID: |
26746328 |
Appl. No.: |
09/135238 |
Filed: |
August 17, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60066063 |
Nov 17, 1997 |
|
|
|
Current U.S.
Class: |
514/44R ;
435/320.1; 435/325; 536/23.1; 536/23.5 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 14/70503 20130101; A61K 38/00 20130101; A61P 37/00 20180101;
A61P 43/00 20180101 |
Class at
Publication: |
514/44 ;
536/23.1; 536/23.5; 435/320.1; 435/325 |
International
Class: |
A61K 048/00; C07H
021/04; C12N 005/08 |
Claims
We claim:
1. A recombinant nucleic acid encoding a Toso protein that will
hybridize under high stringency conditions to the nucleic acid
sequence depicted in FIG. 1 (SEQ ID NO:1) or its complement.
2. A recombinant nucleic acid encoding a Toso protein that is at
least about 70% identical to the amino acid sequence depicted in
FIG. 1 (SEQ ID NO: 1).
3. A recombinant nucleic acid according to claim 2 that is at least
about 70% identical to the nucleic acid sequence depicted in FIG. 1
(SEQ ID NO: 1) or its complement.
4. A recombinant nucleic acid according to claim 1 wherein said
Toso protein is a human Toso protein.
5. A recombinant nucleic acid according to claim 1 encoding the
amino acid sequence depicted in FIG. 1 (SEQ ID NO:1).
6. A recombinant nucleic acid according to claim 1 encoding a Toso
polypeptide that is at least about 70% identical to the sequence of
amino acid residues 18 to 253 of FIG. 2a (SEQ ID NO:2).
7. A recombinant nucleic acid according to claim 1 having at least
70% sequence identity to (a) a DNA molecule encoding a Toso
polypeptide comprising the sequence of amino acid residues 18 to
253 of FIG. 2a (SEQ ID NO:2), or (b) the complement of the DNA
molecule of (a).
8. A recombinant nucleic acid according to claim 1 encoding a Toso
polypeptide that is at least about 70% identical to the sequence of
amino acid residues 18 to 272 of FIG. 2a (SEQ ID NO:2)
9. A recombinant nucleic acid according to claim 1 having at least
70% sequence identity to (a) a nucleic acid molecule encoding a
Toso polypeptide comprising the sequence of amino acid residues 18
to 272 of FIG. 2a (SEQ ID NO:2), or (b) the complement of the
nucleic acid molecule of (a).
10. A recombinant nucleic acid according to claim 1 encoding a Toso
polypeptide that is at least about 70% identical to the sequence of
amino acid residues 273 to 390 of FIG. 2a (SEQ ID NO:2).
11. A recombinant nucleic acid according to claim 1 comprising DNA
having at least 70% sequence identity to (a) a nucleic acid
molecule encoding a Toso polypeptide comprising the sequence of
amino acid residues 273 to 390 of FIG. 2a (SEQ ID NO:2), or (b) the
complement of the nucleic acid molecule of (a).
12. A recombinant nucleic acid according to claim 1 operably linked
to control sequences recognized by a host cell transformed with the
nucleic acid.
13. An expression vector comprising the nucleic acid of claim
12.
14. A host cell comprising the recombinant nucleic acid of claim
1.
15. A host cell comprising the vector of claim 13.
16. A process for producing a Toso protein comprising culturing the
host cell of claim 14 under conditions suitable for expression of a
Toso protein.
17. A process according to claim 16 further comprising recovering
said Toso protein.
18. A Toso protein encoded by a nucleic acid that will hybridize
under high stringency conditions to the complement of the nucleic
acid sequence depicted in FIG. 1 (SEQ ID NO: 1).
19. A recombinant Toso protein that is at least about 70% identical
to the amino acid sequence depicted in FIG. 2a (SEQ ID NO:2).
20. A Toso protein according to claim 18 comprising the sequence
depicted in FIG. 2a (SEQ ID NO:2).
21. A Toso protein according to claim 18 encoded by a nucleic acid
at least about 70% identical to the nucleic acid sequence depicted
in FIG. 1 (SEQ ID NO:1).
22. An isolated polypeptide which specifically binds to a Toso
protein according to claim 18.
23. A polypeptide according to claim 22 that is an antibody.
24. A polypeptide according to claim 23 wherein said antibody is a
monoclonal antibody.
25. A monoclonal antibody according to claim 24 that modulates the
biological function of a Toso protein.
26. A monoclonal antibody according to claim 25 that reduces or
eliminates the biological function of a Toso protein.
27. A monoclonal antibody according to claim 24 that increases the
biological function of a Toso protein.
28. An antibody according to claim 23 directed against the
extracellular domain of the Toso protein comprising the sequence of
amino acid residues 18 to 253 of FIG. 2a (SEQ ID NO:2).
29. An antibody according to claim 23 directed against the
cytoplasmic domain of the Toso protein comprising the sequence of
amino acid residues 273 to 390 of FIG. 2a (SEQ ID NO:2).
30. A method of modulating apoptosis in a cell comprising
administering to said cell a recombinant nucleic acid encoding a
Toso protein.
31. A mammalian cell comprising a modified Toso cell surface
receptor.
32. A method for treating an apoptosis related condition in a
mammal comprising administering a recombinant nucleic acid encoding
a Toso protein.
33. A method for treating an apoptosis related condition in a
mammal comprising administering a Toso protein.
34. A method for treating an apoptosis related condition in a
mammal comprising administering an anti-Toso antibody.
Description
[0001] This is a continuing application of U.S. Ser. No.
60/066,063, filed Nov. 17, 1997.
FIELD OF THE INVENTION
[0002] The invention relates to novel Toso proteins, nucleic acids
and antibodies. The invention further relates to the use of
bioactive agents such as Toso proteins, nucleic acids and
antibodies capable of modulating Fas or tumor necrosis factor
("TNF") receptor mediated apoptosis for the diagnosis and treatment
of disease.
BACKGROUND OF THE INVENTION
[0003] Apoptosis or programmed cell death is an important
homeostatic mechanism that maintains cell number, positioning, and
differentiation. Several intracellular and intercellular processes
are known to regulate apoptosis. One of the best characterized
systems is initiated by the cell surface receptor, Fas
(Apo-1/CD95), homologues of which initiate apoptosis in a wide
range of organisms (Itoh, et. al., Cell, 66:233-243 (1991);
Yonehara, et al., J. Exp. Med., 169:1747-1756 (1989)). Clustering
of the Fas cytoplasmic domain generates an apoptotic signal via the
"death domain" (Itoh and Nagata, J. Biol. Chem., 268:10932-10937
(1993)). Several critical proteins that bind to the death domain or
other domains within the cytoplasmic region have been identified
using yeast two-hybrid and biochemical screens (Boldin, et al., J.
Biol. Chem., 270:7795-7798 (1995); Chinnaiyan, et al., Cell,
8145:505-512 (1995); Chu, et al., Proc. Natl. Acad. Sci. USA,
92:11894-11898 (1995); Okura, et al., J. Immunol., 157:4277-4281
(1996); Sato, et al., Science, 268:411-415 (1995); Stanger, et al,
Cell, 8145:513-523 (1995)).
[0004] Fas engagement by Fas ligand is capable of activating the
interleukin-1 .beta. converting enzyme family of cysteine proteases
(Caspases)--the proteolytic executors of apoptosis (Enari, et al.,
Nature, 375:78-81 (1995); Enari, et al., Nature, 380:723-726
(1996); Los, et al., Nature, 375:81-83 (1995); Tewari and Dixit, J.
Biol. Chem, 270:3255-3260 (1995)). Recent studies implicate
caspase8 (MACH/FLICE/Mch5) as linking Fas receptor signaling to
downstream caspases via its association with FADD/MORT1 (Boldin, et
al., (1995); Chinnaiyan, et al., (1995); Boldin, et al., (1996);
Fernandes-Alnenri, et al., Proc. Natl. Acad Sci. USA, to
93:7464-7469 (1996); Muzio, et al., Cell, 85:817-827 (1996)).
Several groups have reported that caspase-8 activation is inhibited
by a cellular inhibitor, cFLIP/FLAME-1/1-FLICE (Irmler, et al.,
Nature, 388:190-195 (1997); Srinivasula, et al., J. Biol. Chem.,
272:18542-18545 (1997); Hu, et al., J. Biol. Chem., 272:17255-17257
(1997)). Other proteins involved in Fas-mediated apoptosis include:
(a) the Fas-activated serine/threonine kinase (FAST kinase), which
is rapidly activated during Fas-mediated apoptosis; (b) acid
sphingomyelinase, which produces ceramide, a pro-apoptotic signal
that acts as a second messenger in several systems; and (c) Daxx, a
novel protein that links Fas to the JNK stress kinase pathway
(Cifone, et al., J. Exp. Med., 180:1547-1552 (1994); Tian, et al.,
J. Exp. Med., 182:865-874 (1995); Yang, et al., Cell, 89:1067-1076
(1997)). The exact role of these latter co-activators has yet to be
fully defined.
[0005] A balance between life and programmed cell death signals in
cells is likely to be governed by multiple interacting regulators
that counteract apoptotic signals with appropriate anti-apoptotic
signals. Imbalances in this regulation can result in wide variety
of pathologies, including cancer and immune dysftunction and it is
now clear that other polypeptides besides Fas contribute to
disregulation of appropriately induced apoptosis. As an example, in
many tumor cell lines Fas expression does not correlate with
sensitivity to Fas-induced apoptosis, implying the existence of
Fas-resistance protein (Richardson, et al., Eur. J. Immunol.,
24:2640-2645 (1994)). Also, in some types of cells, Fas-induced
apoptosis requires protein synthesis inhibitors such as
cycloheximide (Itoh and Nagata, (1993); Yonehara, et al., (1989))
and even in Fas-sensitive cells, protein synthesis inhibitors can
play a synergistic role with cycloheximide (Itoh and Nagata,
(1993)). These combined observations further suggest the existence
of proteins capable of suppressing Fas-generated apoptotic
signaling.
[0006] Additionally, in the course of a normal immune response,
both cytotoxic T cell and NK cell activation can lead to Fas ligand
(FasL) induction of apoptosis in target cells (Arase, et al., J.
Exp. Med., 181:1235-1238 (1995); Berke, Cell, 81:9-12 (1995);
Montel, et al., Cell Immunol., 166:236-246 (1995)). Although both
Fas and FasL are rapidly induced following T-cell activation,
activated-T cells remain resistant to Fas-induced apoptosis for
several days (Klas, et al., Int. Immunol., 5:625-630 (1993);
Owen-Schaub, et al., Cell Immunol., 140:197-205 (1992)). Thus, a
mechanism exists to shield newly activated T cells from the
cytotoxicity of their own FasL expression. This is likely to be an
important component of T cell activation processes and protection
in Iymph nodes, splenic germinal centers and other sites at which T
cell activation results in apoptosis of target cells.
[0007] Described herein is the identification and characterization
of a novel surface molecule, "Toso" which is a member of the
immunoglobulin gene superfamily and which specifically inhibits Fas
and TNF receptor family mediated apoptosis. The results demonstrate
the existence of cell surface mediated signaling pathways that lead
to down regulation of Fas-mediated apoptosis in certain cell types
and suggest that activation of T cells suppresses internal
signaling systems that might lead inappropriately to T cell-induced
self-killing.
[0008] Accordingly, it is an object of the invention to provide
Toso proteins and related molecules. It is a further object of the
invention to provide recombinant nucleic acids encoding Toso
proteins, and expression vectors and host cells containing the
nucleic acid encoding the Toso protein. A further object of the
invention is to provide methods for screening for antagonists and
agonists of Toso.
SUMMARY OF THE INVENTION
[0009] In accordance with the objects outlined above, the present
invention provides recombinant nucleic acids encoding a Toso
protein that will hybridize under high stringency conditions to the
nucleic acid sequence depicted in FIG. 1 (SEQ ID NO:1) or its
complement. Recombinant nucleic acids encoding Toso proteins that
are at least about 70% identical to the amino acid sequence
depicted in FIG. 1 (SEQ ID NO:1) are also provided, as well as
recombinant nucleic acid that is at least about 70% identical to
the nucleic acid sequence depicted in FIG. 1 (SEQ ID NO: 1) or its
complement.
[0010] In a further aspect, the invention provides expression
vectors and host cells comprising the nucleic acids of the
invention, and processes for producing a Toso protein comprising
culturing the host cells under conditions suitable for expression
of a Toso protein.
[0011] In an additional aspect, the invention provides Toso
proteins, and antibodies that bind to Toso proteins.
[0012] Further provided are methods of modulating apoptosis in a
cell comprising administering to the cell a recombinant nucleic
acid encoding a Toso protein, and methods for treating an apoptosis
related condition in a mammal comprising administering a
recombinant nucleic acid encoding a Toso protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 depicts the nucleotide sequence (SEQ ID: NO 1) of
Toso. Also presented are the positions of the initiator ATG start
codon, the stop codon the nucleotides which correspond to the
signal sequence and the nucleotides which correspond to the
putative transmembrane domain of the Toso protein.
[0014] FIG. 2a depicts the amino acid sequence (SEQ ID:NO 2) of
amino acids 1 to 390 deduced from nucleotides 1 to 1173 of the
nucleotide sequence shown in FIG. 1 (SEQ ID:NO 1). Two hydrophobic
regions are underlined.
[0015] FIG. 2b depicts a Kyte-Dolittle hydropathy plot analysis of
Toso gene product (upper) and schematic presentation of Toso
(bottom). The mature Toso is a 390-amino acid protein with the
leader sequence of 17 amino acids (hatched bar), the extracellular
domain of 236 amino acids (ED), the transmembrane region of 20
amino acids (TM; dotted bar) and the cytoplasmic domain of 117
amino acids (CD). The immunoglobulin domain (Ig), the basic amino
acid-rich region (Basic), the proline-rich region (Proline), and
the acidic amino acid-rich region (Acidic) are indicated.
[0016] FIG. 3 depicts BLAST search results using the Toso gene
product. The position of the first amino acid in each sequence is
given in the left side of the alignment. Gaps are indicated by
dashes. Dark and light shading refer to identical and similar
residues, respectively. For sequence alignment of the Toso
N-terminus, IgVH (G1 HUNM), IgV.lambda. (L 1 MS4E), TcR V.alpha.
(RWMSAV), TCR V.beta. (RWHUVY), CD4 (U47924), CD8 chain 11
(X04310), Poly Ig R (QRRBG) and immunoglobulin V-set consensus
sequence are shown in the alignment. Arrows indicate positions
characteristic of many V-set sequences. The sequence of the Toso
cytoplasmic domain is aligned with acid sphingomyelinase, insulin
receptor substrate 1 (IRS1) and apoptosis inhibitor, IAP, from
Orgyia pseudotsugata nuclear polyhedrosis virus (Op-1AP).
[0017] FIG. 4a depicts the effect of Toso on anti-Fas induced
apoptosis. The percentage of apoptotic cells are expressed as the
mean (hatched and shaded bar) .+-.SD of triplicate cultures.
Apoptotic cells in each culture without anti-Fas mAb were less than
2%.
[0018] FIG. 4b depicts the effect of Toso on anti-Fas-,
staurosporine- and ceramide-induced apoptosis in Jurkat.ecoR cells
(closed triangle), Jurkat.ecoR cells infected with pBabeMN-lacZ
(closed square) and pBabeMN-Toso (open circle). The percentage of
apoptotic cells are expressed as the mean (symbol) .+-.SD (vertical
bar) of triplicate cultures.
[0019] FIG. 4c depicts the effect of Toso on FADD-induced apoptosis
in Jurkat.ecoR cells infected with pBabeMN-Lyt-2-.alpha.' (hatched
bar), and pBabeMN-Toso (shaded bar). The percentage of apoptotic
cells are expressed as the mean (hatched bar or shaded bar) .+-.SD
of triplicate cultures.
[0020] FIG. 4d depicts the effect of Toso on TNF-.alpha.-induced
apoptosis in Jurkat.ecoR cells. The percentage of apoptotic cells
are expressed as the mean (hatched bar or shaded bar) .+-.SD of
triplicate cultures.
[0021] FIG. 4e depicts the effect Toso on anti-Fas mAb-induced
apoptosis in cells cultured with (.alpha.-Fas (+)) or without
(.alpha.-Fas (-)) 50 ng/ml of anti-Fas mAb. After culture for five
days, GFP expression of survived cells were analyzed by
FACScan.
[0022] FIG. 5a depicts the results of Western blot analysis of
caspase-8 processing by induction of cFLIP. Jurkat.ecoR cells
(control) and pBabeMN-Toso-infected Jurkat.ecoR cells (Toso) were
cultured with (+) or without (-) 50 ng/ml of anti-Fas mAb
(.alpha.-Fas) for 6 hours. Positions of pro-caspase-8 (Pro), the
processed form (p20) and standard marker are indicated.
[0023] FIG. 5b depicts the results of Western blot analysis of
cFLIP expression in Jurkat.ecoR cells (control) and
pBabeMN-Toso-infected Jurkat.ecoR cells (Toso).
[0024] FIG. 6a depicts the effect of Toso deletion mutant
expression on anti-Fas mAb-induced apoptosis. Structure of the Toso
deletion mutants is shown at the left side of this panel.
Full-length Toso is a 390-amino acid protein with the leader
sequence of 17 amino acids (hatched bar), the extracellular domain
of 236 amino acids (ED), the transmembrane region of 20 amino acids
(TM; dark-shaded bar) and the cytoplasmic domain of 117 amino acids
(CD).
[0025] The hemagglutinin (HA) tag is indicated as a light shaded
bar. The percentage of apoptotic cells is expressed as the mean
(hatched and shaded bar) .+-.SD of triplicate cultures.
[0026] FIG. 6b depicts Western blot analysis of deletion mutants
using anti-HA antibody. The molecular weight of major products from
Toso.HA, Toso.DELTA. (377-390).HA, Toso.DELTA.(334-390). HA,
Toso.DELTA.(252-390). HA, Toso.DELTA.(281-390). HA,
Toso.DELTA.(291-187). HA and Lyt-2/Toso(271-390).HA was 60/35,
55/30, 50/26, 40, 38, 35, 60/30 kDa, respectively. Positions and
sizes (kDa) of standard protein markers are indicated in left side
of panel.
[0027] FIG. 6c depicts Crosslinking the extracellular domain of
Toso. Positions of standard protein markers and Toso.HA are
indicated in left side and right of panel, respectively.
[0028] FIG. 7a depicts mRNA dot blot analysis of Toso gene in
several human tissues.
[0029] FIG. 7b depicts Northern blot analysis of Toso gene in
several human immune tissues. Positions and sizes (kbp) of Toso
mRNA are indicated in left side of panels.
[0030] FIG. 7c depicts RT-PCR analysis of Toso in human cell lines
(upper panel). Positions and sizes (kbp) of Toso and standard
nucleotide makers are indicated. As a control for loading, we
amplified .beta.-actin cDNA (lower panels).
[0031] FIG. 8a depicts (a) Northern blot analysis of Toso gene in
Jurkat cells (None) and Jurkat cells stimulated with PMA and PHA
(PMA+PHA) or PMA and lonomycin (PMA+lo.). RNA was electrophoresed,
transferred to a Hybond N+ membrane and hybridized with a
radiolabelled probe specific for Toso (upper) and .beta.-actin
(lower). Film was exposed at -70.degree. C. with an intensifying
screen for two days (upper). Positions and sizes (kbp) of Toso mRNA
are indicated in right side of panels.
[0032] FIG. 8b depicts activation induced resistance to anti-Fas
mAb-induced apoptosis in Jurkat cells. The percentage of apoptotic
cells are expressed as the mean (hatched bar) .+-.SD of triplicate
cultures.
[0033] FIG. 8c depicts the effect of Toso on PMA and lonomycin
(PMA+lo.)-induced apoptosis. Jurkat.ecoR cells (-), Jurkat.ecoR
cells infected with pBabeMN-lacZ (lacZ), pBabeMN-Toso-infected
clones (Toso clones 1-5) were cultured with 10 ng/ml of anti-Fas
mAb (left), 10 ng/ml PMA and 500 ng/ml lonomycin (right) for 24
hours. The percentage of apoptotic cells are expressed as the mean
(hatched bar and shaded bar) +SD of triplicate cultures.
[0034] FIG. 9a depicts the RT-PCR analysis of Toso in peripheral
blood mononuclear cells after activation with PHA (upper panel, the
1.2 kbp fragment of Toso).
[0035] FIG. 9b depicts analysis of Toso in peripheral blood
mononuclear cells after allogenic stimulation (upper panel, the 1.2
kbp fragment of Toso). Stimulator cells (SC), responder cells (RC)
or mixed cells (RC+SC) were cultured for one day (day 1) and six
days (day 6).
[0036] FIG. 10 depicts a model for the role of Toso in T cell
activation. In the model, the role of Toso is to be induced
following T cell activation and to protect T cells from
self-induced programmed cell death. The inhibitory effects of Toso
on Fas signaling maps at the level of caspase-8 through induced
expression of cFLIP.
[0037] FIG. 11 depicts massive cell death of 70Z/3 cells induced by
TOSO. 70Z/3 cells were incubated with supernatant from .phi.NX-E
(closed triangle), viral supernatant of pBabeMN-Lyt-2 (closed
square), or pBabeMN-TOSO (open circle) for 12 hours including the
initial spinning at 2500 rpm for 90 min. Infection frequency of
pBabeMN-Lyt-2 was determined to be 79% at 48 hours post infection.
The percentage of viable cells at various time points are expressed
as mean (symbol) .+-.SD (vertical bar) of triplicate cultures.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention provides novel Ig domain-containing
Toso polypeptides, with potent pathway-specific anti-apoptotic
effects in hematopoietic cells. Toso (named after a Japanese liquor
that is drunk on New Year's Day to celebrate long life and eternal
youth) exerts an inhibitory activity against apoptosis induced by
Fas-, TNF-.alpha.-, FADD and PMA/lonomycin but not against
staurosporine- or ceramide-induced apoptosis. Without being bound
by theory, the mechanism of blocking apoptotic activation, and the
pathway specificity of the effect, is most likely explained by Toso
induction of cFLIP expression which inhibits caspase-8 processing.
Toso is expressed within lymphoid tissues and hematopoietic cells,
and is enhanced after T-cell activation, which suggests an
important role for this and related molecules in the immune
system.
[0039] Accordingly, the present invention provides Toso proteins
and nucleic acids. In a preferred embodiment, the Toso proteins are
from vertebrates and more preferably from mammals including dogs,
cats and rabbits, rodents (including rats, mice, hamsters, guinea
pigs, etc.), primates (including chimpanzees, African green
monkeys, etc.), farm animals (including sheep, goats, pigs, cows,
horses, etc.) and in the most preferred embodiment, from humans.
However, using the techniques outlined below, Toso proteins from
other organisms may also be obtained.
[0040] As outlined herein, the Toso proteins of the present
invention are IgG superfamily molecules which are expressed in a
variety of tissue types, including, but not limited to lymph nodes,
peripheral blood leukocytes, thymus, lung, and kidney. As further
outlined herein, Toso proteins exert pathway specific
anti-apoptotic effects in hematopoietic cells. Toso is a membrane
bound protein, as it contains a putative transmembrane domain. The
extracellular domain of Toso has homology to immunoglobulin
variable domains.
[0041] A Toso protein of the present invention may be identified in
several ways. "Protein" in this sense includes proteins,
polypeptides, and peptides. A Toso nucleic acid or Toso protein is
initially identified by substantial nucleic acid and/or amino acid
sequence homology to the sequences shown in FIGS. 1 and 2a. Such
homology can be based upon the overall nucleic acid or amino acid
sequence.
[0042] As used herein, a protein is a "Toso protein" if the overall
homology of the protein sequence to the amino acid sequence shown
in FIG. 2a (SEQ ID NO:2) is preferably greater than about 50 or
60%, more preferably greater than about 70 or 75%, even more
preferably greater than about 80% and most preferably greater than
85%. In some embodiments the homology will be as high as about 90
to 95 or 98%. Homology in this context means sequence similarity or
identity, with identity being preferred. Identical in this context
means identical amino acids at corresponding positions in the two
sequences which are being compared. Homology in this context
includes amino acids which are identical and those which are
similar (functionally equivalent). This homology will be determined
using standard techniques known in the art, such as the Best Fit
sequence program described by Devereux, et al., Nucl. Acid Res.
12:387-395 (1984), preferably using the default settings, or the
BLASTX program (Altschul, et al., J. Mol. Biol., 215:403-410
(1990)). The alignment may include the introduction of gaps in the
sequences to be aligned. In addition, for sequences which contain
either more or fewer amino acids than the proteins shown in the
Figures, it is understood that the percentage of homology will be
determined based on the number of homologous amino acids in
relation to the total number of amino acids. Thus, for example,
homology of sequences shorter than that shown in the Figures, as
discussed below, will be determined using the number of amino acids
in the shorter sequence.
[0043] As outlined herein, Toso proteins have several important
domains. Toso contains a cytoplasmic domain from amino acids 273 to
390, with the extracellular domain spanning from 18 to 253 (unless
otherwise specified, all amino acid numbering is based on the human
sequence). Toso contains a standard transmembrane domain, spanning
from amino acids 254 to 272. Toso contains an additional
hydrophobic region at the N-terminus, amino acids 1 to 17,
corresponding to a putative signal sequence. In addition, the
cytoplasmic domain of Toso contains a basic amino acid-rich region
(from Arg274 to Arg323), a proline rich region (from Pro334 to
P346), and an acidic amino acid-rich region (from Glu378 to
Asp384). In addition, the cytoplasmic domain has partial homology
to FAST kinase, acid sphingomyleinase, insulin receptor substrate-1
(IRS-1) and the apoptosis inhibitor from Orgyia pseudotsugata
nuclear polyhedrosis virus (Op-1AP). The extracellular domain of
Toso has homology to the immunoglobulin V-region.
[0044] As used herein, a protein is also a "Toso protein" if the
homology of the cytoplasmic domain comprising amino acids 273 to
390, or the extracellular domain comprising amino acids 18 to 253,
respectively, of the amino acid sequence shown in FIG. 2a (SEQ ID
NO:2) is preferably greater than about 50% of 60%, more preferably
greater than about 70% or 75%, even more preferably greater than
about 80% and most preferably greater than 85%. In some embodiments
the homology will be as high as about 90 to 95 or 98%.
[0045] Toso proteins of the present invention may be shorter or
longer than the amino acid sequences shown in the Figures. Thus, in
a preferred embodiment, included within the definition of Toso
proteins are portions or fragments of the sequences depicted in the
Figures.
[0046] As outlined herein, Toso deletion mutants can be made,
including, but not limited to, the deletion of amino acids 377-390,
334-390, 281-390, 252-390, and 29-187. As further outlined herein,
Toso fusion proteins can be made including, but not limited to, the
fusion of amino acids 1-271. A preferred Toso fragment is the
cytoplasmic domain of Toso, which may modulate apoptosis, as shown
herein. A further preferred Toso fragment is the extracellular
domain of Toso, comprising roughly the first 236 amino acids of
Toso, which is required for the anti-apoptotic effects on Fas
antibody-stimulated cells. However, as outlined herein, preferred
fragments of Toso also include a transmembrane domain, as it may be
involved in signaling and Fas-induced apoptosis by Toso may require
its insertion into membranes.
[0047] Thus, in a preferred embodiment, the Toso proteins of the
present invention are Toso polypeptides. In this embodiment, a Toso
polypeptide comprises at least the immunoglobin V-like domain, and
preferably a transmembrane domain, although it may contain
additional amino acids as well. As shown in the Examples and
discussed below, Toso is an IgG superfamily protein which is
capable of inhibiting apoptosis mediated by members of the Fas or
TNF receptor family of proteins.
[0048] In a preferred embodiment, the Toso proteins are derivative
or variant Toso proteins. That is, as outlined more fully below,
the derivative Toso peptide will contain at least one amino acid
substitution, deletion or insertion, with amino acid substitutions
being particularly preferred. The amino acid substitution,
insertion or deletion may occur at any residue within the Toso
peptide. As outlined below, particularly preferred substitutions
are made within the extracellular domain or cytoplasmic domain of
the Toso protein.
[0049] In addition, as is more fully outlined below, Toso proteins
can be made that are longer than those depicted in the figures, for
example, by the addition of epitope or purification tags, the
addition of other fusion sequences, etc.
[0050] Toso proteins may also be identified as being encoded by
Toso nucleic acids. Thus, Toso proteins are encoded by nucleic
acids that will hybridize to the sequence depicted in FIG. 1, or
its complement, as outlined herein.
[0051] In a preferred embodiment, when the Toso protein is to be
used to generate antibodies, the Toso protein must share at least
one epitope or determinant with the full length protein shown in
FIG. 2a. By "epitope" or "determinant" herein is meant a portion of
a protein which will generate and/or bind an antibody. Thus, in
most instances, antibodies made to a smaller Toso protein will be
able to bind to the full length protein. In a preferred embodiment,
the epitope is unique; that is, antibodies generated to a unique
epitope show little or no cross-reactivity. In a preferred
embodiment, the antibodies are generated to an extracellular
portion of the Toso molecule, i.e. to all or some of the N-terminal
region from amino acid numbers 18-253.
[0052] In a preferred embodiment, the antibodies to Toso are
capable of reducing or eliminating the biological function of Toso,
as is described below. That is, the addition of anti-Toso
antibodies (either polyclonal or preferably monoclonal) to cells
comprising Toso receptors may reduce or eliminate the Toso receptor
activity, blocking the signalling pathway that blocks apoptosis;
that is, when Toso receptor function is reduced or eliminated, the
cells die. Generally, at least a 50% decrease in activity is
preferred, with at least about 75% being particularly preferred and
about a 95-100% decrease being especially preferred.
[0053] The Toso antibodies of the invention specifically bind to
Toso proteins. By "specifically bind" herein is meant that the
antibodies bind to the protein with a binding constant in the range
of at least 10.sup.6-10.sup.8M, with a preferred range being
10.sup.7- 10.sup.9M.
[0054] In the case of the nucleic acid, the overall homology of the
nucleic acid sequence is commensurate with amino acid homology but
takes into account the degeneracy in the genetic code and codon
bias of different organisms. Accordingly, the nucleic acid sequence
homology may be either lower or higher than that of the protein
sequence. Thus the homology of the nucleic acid sequence as
compared to the nucleic acid sequence of FIG. 1 is preferably
greater than 50 or 60%, more preferably greater than about 70 to
75%, particularly greater than about 80% and most preferably
greater than 85%. In some embodiments the homology will be as high
as about 90 to 95 or 98%.
[0055] In a preferred embodiment, a Toso nucleic acid encodes a
Toso protein. As will be appreciated by those in the art, due to
the degeneracy of the genetic code, an extremely large number of
nucleic acids may be made, all of which encode the Toso proteins of
the present invention. Thus, having identified a particular amino
acid sequence, those skilled in the art could make any number of
different nucleic acids, by simply modifying the sequence of one or
more codons in a way which does not change the amino acid sequence
of the Toso.
[0056] In one embodiment, the nucleic acid homology is determined
through hybridization studies. Thus, for example, nucleic acids
which hybridize under high stringency to the nucleic acid sequences
shown in FIG. 1 or its complement is considered a Toso gene. High
stringency conditions are known in the art; see for example
Maniatis, et al., Molecular Cloning: A Laboratory Manual, 2d
Edition (1989), and Short Protocols in Molecular Biology, ed.
[0057] Ausubel, et al., both of which are hereby incorporated by
reference. [An example of such conditions includes hybridization at
about 65.degree. C. in about 5.times. SSPE and washing conditions
of about 65.degree. C. in about 0.1.times. SSPE.
[0058] In another embodiment, less stringent hybridization
conditions are used; for example, moderate or low stringency
conditions may be used, as are known in the art; see Maniatis and
Ausubel, supra. An example of such conditions includes
hybridization at about 50 to 55.degree. C. in 5.times. SSPE and
washing conditions of about 50.degree. C. in about 5.times.
SSPE.
[0059] The Toso proteins and nucleic acids of the present invention
are preferably recombinant. As used herein, "nucleic acid" may
refer to either DNA or RNA, or molecules which contain both deoxy-
and ribonucleotides. The nucleic acids include genomic DNA, cDNA
and oligonucleotides including sense and anti-sense nucleic acids.
Such nucleic acids may also contain modifications in the
ribose-phosphate backbone to increase stability and half life of
such molecules in physiological environments.
[0060] The nucleic acid may be double stranded, single stranded, or
contain portions of both double stranded or single stranded
sequence. As will be appreciated by those in the art, the depiction
of a single strand ("Watson") also defines the sequence of the
other strand ("Crick"); thus the sequence depicted in FIG. 1 also
includes the complement of the sequence. By the term "recombinant
nucleic acid" herein is meant nucleic acid, originally formed in
vitro, in general, by the manipulation of nucleic acid by
endonucleases, in a form not normally found in nature. Thus an
isolated Toso nucleic acid, in a linear form, or an expression
vector formed in vitro by ligating DNA molecules that are not
normally joined, are both considered recombinant for the purposes
of this invention. It is understood that once a recombinant nucleic
acid is made and reintroduced into a host cell or organism, it will
replicate non-recombinantly, i.e. using the in vivo cellular
machinery of the host cell rather than in vitro manipulations;
however, such nucleic acids, once produced recombinantly, although
subsequently replicated non-recombinantly, are still considered
recombinant for the purposes of the invention.
[0061] Similarly, a "recombinant protein" is a protein made using
recombinant techniques, i.e. through the expression of a
recombinant nucleic acid as depicted above. A recombinant protein
is distinguished from naturally occurring protein by at least one
or more characteristics. For example, the protein may be isolated
or purified away from some or all of the proteins and compounds
with which it is normally associated in its wild type host, and
thus may be substantially pure. For example, an isolated protein is
unaccompanied by at least some of the material with which it is
normally associated in its natural state, preferably constituting
at least about 0.5%, more preferably at least about 5% by weight of
the total protein in a given sample. A substantially pure protein
comprises at least about 75% by weight of the total protein, with
at least about 80% being preferred, and at least about 90% being
particularly preferred. The definitiin includes the production of a
Toso protein from one organism in a different organism or host
cell. Alternatively, the protein may be made at a significantly
higher concentration than is normally seen, through the use of a
inducible promoter or high expression promoter, such that the
protein is made at increased concentration levels. Alternatively,
the protein may be in a form not normally found in nature, as in
the addition of an epitope tag or amino acid substitutions,
insertions and deletions, as discussed below.
[0062] Once identified, the polypeptides comprising the
biologically active sequences may be prepared in accordance with
conventional techniques, such as synthesis (for example, use of a
Beckman Model 990 peptide synthesizer or other commercial
synthesizer).
[0063] Also included within the definition of Toso proteins of the
present invention are amino acid sequence variants. These variants
fall into one or more of three classes: substitutional, insertional
or deletional variants. These variants ordinarily are prepared by
site specific mutagenesis of nucleotides in the DNA encoding the
Toso protein, using cassette or PCR mutagenesis or other techniques
well known in the art, to produce DNA encoding the variant, and
thereafter expressing the DNA in recombinant cell culture as
outlined above. However, variant Toso protein fragments having up
to about 100-150 residues may be prepared by in vitro synthesis
using established techniques. Amino acid sequence variants are
characterized by the predetermined nature of the variation, a
feature that sets them apart from naturally occurring allelic or
interspecies variation of the Toso protein amino acid sequence. The
variants typically exhibit the same qualitative biological activity
as the naturally occurring analogue, although variants can also be
selected which have modified characteristics as will be more fully
outlined below.
[0064] While the site or region for introducing an amino acid
sequence variation is predetermined, the mutation per se need not
be predetermined. For example, in order to optimize the performance
of a mutation at a given site, random mutagenesis may be conducted
at the target codon or region and the expressed Toso variants
screened for the optimal combination of desired activity.
Techniques for making substitution mutations at predetermined sites
in DNA having a known sequence are well known, for example, Ml 3
primer mutagenesis and PCR mutagenesis. Screening of the mutants is
done using assays of Toso protein activities; for example, for
binding domain mutations, competitive binding studies such as are
outlined in the Examples may be done.
[0065] Amino acid substitutions are typically of single residues;
insertions usually will be on the order of from about 1 to 20 amino
acids, although considerably larger insertions may be tolerated.
Deletions range from about 1 to about 20 residues, although in some
cases deletions may be much larger. For example, a preferred
variant comprises the deletion of the cytoplasmic domain, leaving
only the extracellular domain of Toso, preferably including the
transmembrane domain. Additional preferred variants comprise the
cytoplasmic domain alone or a soluble receptor (i.e. the
extracellular domain lacking the transmembrane domain).
[0066] Substitutions, deletions, insertions or any combination
thereof may be used to arrive at a final derivative. Generally
these changes are done on a few amino acids to minimize the
alteration of the molecule. However, larger changes may be
tolerated in certain circumstances. When small alterations in the
characteristics of the Toso protein are desired, substitutions are
generally made in accordance with the following chart:
1 Chart I Original Residue Exemplary Substitutions Ala Ser Arg Lys
Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln
Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met,
Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu
[0067] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those shown in Chart I. For example, substitutions may be made
which more significantly affect: the structure of the polypeptide
backbone in the area of the alteration, for example the
alpha-helical or beta-sheet structure; the charge or hydrophobicity
of the molecule at the target site; or the bulk of the side chain.
The substitutions which in general are expected to produce the
greatest changes in the polypeptide's properties are those in which
(a) a hydrophilic residue, e.g. seryl or threonyl, is substituted
for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is
substituted for (or by) any other residue; (c) a residue having an
electropositive side chain, e.g., lysyl, arginyl, or histidyl, is
substituted for (or by) an electronegative residue, e.g., glutamyl
or aspartyl; or (d) a residue having a bulky side chain, e.g.,
phenylalanine, is substituted for (or by) one not having a side
chain, e.g., glycine.
[0068] The variants typically exhibit the same qualitative
biological activity and will elicit the same immune response as the
naturally-occurring analogue, although variants also are selected
to modify the characteristics of the Toso proteins as needed.
Alternatively, the variant may be designed such that the biological
activity of the Toso protein is altered. For example, glycosylation
sites, and more particularly one or more O-linked or N-linked
gylcosylation sites may be altered or removed. Either or both of
the transmembrane domains may be altered or removed, to make a
soluble or secreted protein, i.e. the extracellular domain.
[0069] Covalent modifications of Toso polypeptides are included
within the scope of this invention. One type of covalent
modification includes reacting targeted amino acid residues of a
Toso polypeptide with an organic derivatizing agent that is capable
of reacting with selected side chains or the N-or C-terminal
residues of a Toso polypeptide. Derivatization with bifunctional
agents is useful, for instance, for crosslinking Toso to a
water-insoluble support matrix or surface for use in the method for
purifying anti-Toso antibodies or screening assays, as is more
fully described below. Commonly used crosslinking agents include,
e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl- )dithio]propioimidate.
[0070] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the "-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0071] Another type of covalent modification of the Toso
polypeptide included within the scope of this invention comprises
altering the native glycosylation pattern of the polypeptide.
"Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence Toso polypeptide, and/or adding one or
more glycosylation sites that are not present in the native
sequence Toso polypeptide.
[0072] Addition of glycosylation sites to Toso polypeptides may be
accomplished by altering the amino acid sequence thereof. The
alteration may be made, for example, by the addition of, or
substitution by, one or more serine or threonine residues to the
native sequence Toso polypeptide (for O-linked glycosylation
sites). The Toso amino acid sequence may optionally be altered
through changes at the DNA level, particularly by mutating the DNA
encoding the Toso polypeptide at preselected bases such that codons
are generated that will translate into the desired amino acids.
[0073] Another means of increasing the number of carbohydrate
moieties on the Toso polypeptide is by chemical or enzymatic
coupling of glycosides to the polypeptide. Such methods are
described in the art, e.g., in WO 87/05330 published Sep. 11, 1987,
and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981).
[0074] Removal of carbohydrate moieties present on the Toso
polypeptide may be accomplished chemically or enzymatically or by
mutational substitution of codons encoding for amino acid residues
that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and described, for instance, by
Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by
Edge, et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties on polypeptides can be achieved by the use of
a variety of endo-and exo-glycosidases as described by Thotakura,
et al., Meth. Enzymol., 138:350 (1987).
[0075] Another type of covalent modification of Toso comprises
linking the Toso polypeptide to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat.
Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or
4,179,337.
[0076] Toso polypeptides of the present invention may also be
modified in a way to form chimeric molecules comprising a Toso
polypeptide fused to another, heterologous polypeptide or amino
acid sequence. In one embodiment, such a chimeric molecule
comprises a fusion of a Toso polypeptide with a tag polypeptide
which provides an epitope to which an anti-tag antibody can
selectively bind. The epitope tag is generally placed at the
amino-or carboxyl-terminus of the Toso polypeptide. The presence of
such epitope-tagged forms of a Toso polypeptide can be detected
using an antibody against the tag polypeptide. Also, provision of
the epitope tag enables the Toso polypeptide to be readily purified
by affinity purification using an anti-tag antibody or another type
of affinity matrix that binds to the epitope tag. In an alternative
embodiment, the chimeric molecule may comprise a fusion of a Toso
polypeptide with an immunoglobulin or a particular region of an
immunoglobulin. For a bivalent form of the chimeric molecule, such
a fusion could be to the Fc region of an IgG molecule or GST
fusions.
[0077] Various tag polypeptides and their respective antibodies are
well known in the art. Examples include poly-histidine (poly-his)
or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 [Field, et al., Mol. Cell Biol.,
8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7,
and 9E10 antibodies thereto [Evan, et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody [Paborsky, et al., Protein
Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include
the Flag-peptide [Hopp, et al., Biol. echnology, 6:1204-1210
(1988)]; the KT3 epitope peptide [Martin, et al., Science,
255:192-194 (1992)]; tubulin epitope peptide [Skinner, et al., J.
Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein
peptide tag [Lutz-Freyermuth, et al., Proc. Natl. Acad. Sci. USA,
87:6393-6397 (1990)].
[0078] Also included with the definition of Toso protein are other
Toso proteins of the Toso family, and Toso proteins from other
organisms, which are cloned and expressed as outlined below. Thus,
probe or degenerate polymerase chain reaction (PCR) primer
sequences may be used to find other related Toso proteins from
humans or other organisms. As will be appreciated by those in the
art, particularly useful probe and/or PCR primer sequences include
the unique areas of the Toso nucleic acid sequence. Thus, useful
probe or primer sequences may be designed to: all or part of the
sequence of the immunoglobulin V-like Toso domain, all or part of
the unique extracellular domain, which spans roughly amino acids
18-253, or sequences outside the coding region. As is generally
known in the art, preferred PCR primers are from about 15 to about
35 nucleotides in length, with from about 20 to about 30 being
preferred, and may contain inosine as needed. The conditions for
the PCR reaction are well known in the art.
[0079] Once the Toso nucleic acid is identified, it can be cloned
and, if necessary, its constituent parts recombined to form the
entire Toso nucleic acid. Once isolated from its natural source,
e.g., contained within a plasmid or other vector or excised
therefrom as a linear nucleic acid segment, the recombinant Toso
nucleic acid can be further-used as a probe to identify and isolate
other Toso nucleic acids. It can also be used as a "precursor"
nucleic acid to make modified or variant Toso nucleic acids and
proteins.
[0080] Using the nucleic acids of the present invention which
encode a Toso protein, a variety of expression vectors are made.
The expression vectors may be either self-replicating
extrachromosomal vectors or vectors which integrate into a host
genome. Generally, these expression vectors include transcriptional
and translational regulatory nucleic acid operably linked to the
nucleic acid encoding the Toso protein. The term "control
sequences" refers to DNA sequences necessary for the expression of
an operably linked coding sequence in a particular host organism.
The control sequences that are suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize
promoters, polyadenylation signals, and enhancers.
[0081] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice. The transcriptional and
translational regulatory nucleic acid will generally be appropriate
to the host cell used to express the Toso protein; for example,
transcriptional and translational regulatory nucleic acid sequences
from Bacillus are preferably used to express the Toso protein in
Bacillus. Numerous types of appropriate expression vectors, and
suitable regulatory sequences are known in the art for a variety of
host cells.
[0082] In general, the transcriptional and translational regulatory
sequences may include, but are not limited to, promoter sequences,
ribosomal binding sites, transcriptional start and stop sequences,
translational start and stop sequences, and enhancer or activator
sequences. In a preferred embodiment, the regulatory sequences
include a promoter and transcriptional start and stop
sequences.
[0083] Promoter sequences encode either constitutive or inducible
promoters. The promoters may be either naturally occurring
promoters or hybrid promoters. Hybrid promoters, which combine
elements of more than one promoter, are also known in the art, and
are useful in the present invention.
[0084] In addition, the expression vector may comprise additional
elements. For example, the expression vector may have two
replication systems, thus allowing it to be maintained in two
organisms, for example in mammalian or insect cells for expression
and in a procaryotic host for cloning and amplification.
Furthermore, for integrating expression vectors, the expression
vector contains at least one sequence homologous to the host cell
genome, and preferably two homologous sequences which flank the
expression construct. The integrating vector may be directed to a
specific locus in the host cell by selecting the appropriate
homologous sequence for inclusion in the vector. Constructs for
integrating vectors are well known in the art.
[0085] In addition, in a preferred embodiment, the expression
vector contains a selectable marker gene to allow the selection of
transformed host cells. Selection genes are well known in the art
and will vary with the host cell used.
[0086] A preferred expression vector system is a retroviral vector
system such as is generally described in PCT/US97/01019 and
PCT/US97/01048, both of which are hereby expressly incorporated by
reference.
[0087] The Toso proteins of the present invention are produced by
culturing a host cell transformed with an expression vector
containing nucleic acid encoding a Toso protein, under the
appropriate conditions to induce or cause expression of the Toso
protein. The conditions appropriate for Toso protein expression
will vary with the choice of the expression vector and the host
cell, and will be easily ascertained by one skilled in the art
through routine experimentation. For example, the use of
constitutive promoters in the expression vector will require
optimizing the growth and proliferation of the host cell, while the
use of an inducible promoter requires the appropriate growth
conditions for induction. In addition, in some embodiments, the
timing of the harvest is important. For example, the baculoviral
systems used in insect cell expression are lytic viruses, and thus
harvest time selection can be crucial for product yield.
[0088] Appropriate host cells include yeast, bacteria,
archebacteria, fungi, and insect and animal cells, including
mammalian cells, for example primary cells, including stem cells,
including, but not limited to bone marrow stem cells. Of particular
interest are Drosophila melangaster cells, Saccharomyces cerevisiae
and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129
cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells,
fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid
and lymphoid cell lines, Jukat cells, human cells and other primary
cells.
[0089] In a preferred embodiment, the Toso proteins are expressed
in mammalian cells. Mammalian expression systems are also known in
the art, and include retroviral systems. A mammalian promoter is
any DNA sequence capable of binding mammalian RNA polymerase and
initiating the downstream (3') transcription of a coding sequence
for Toso protein into mRNA. A promoter will have a transcription
initiating region, which is usually placed proximal to the 5' end
of the coding sequence, and a TATA box, using a located 25-30 base
pairs upstream of the transcription initiation site. The TATA box
is thought to direct RNA polymerase II to begin RNA synthesis at
the correct site. A mammalian promoter will also contain an
upstream promoter element (enhancer element), typically located
within 100 to 200 base pairs upstream of the TATA box. An upstream
promoter element determines the rate at which transcription is
initiated and can act in either orientation. Of particular use as
mammalian promoters are the promoters from mammalian viral genes,
since the viral genes are often highly expressed and have a broad
host range. Examples include the SV40 early promoter, mouse mammary
tumor virus LTR promoter, adenovirus major late promoter, herpes
simplex virus promoter, the CMV promoter, a retroviral LTR
promoter, mouse maloney luekemia virus LTR, or pBabeMN.
[0090] Typically, transcription termination and polyadenylation
sequences recognized by mammalian cells are regulatory regions
located 3' to the translation stop codon and thus, together with
the promoter elements, flank the coding sequence. The 3' terminus
of the mature mRNA is formed by site-specific post-translational
cleavage and polyadenylation. Examples of transcription terminator
and polyadenlytion signals include those derived form SV40.
[0091] The methods of introducing exogenous nucleic acid into
mammalian hosts, as well as other hosts, is well known in the art,
and will vary with the host cell used. Techniques include
dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, protoplast fusion,
electroporation, viral infection, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the
DNA into nuclei.
[0092] In a preferred embodiment, Toso proteins are expressed in
bacterial systems. Bacterial expression systems are well known in
the art.
[0093] A suitable bacterial promoter is any nucleic acid sequence
capable of binding bacterial RNA polymerase and initiating the
downstream (3') transcription of the coding sequence of Toso
protein into mRNA. A bacterial promoter has a transcription
initiation region which is usually placed proximal to the 5' end of
the coding sequence. This transcription initiation region typically
includes an RNA polymerase binding site and a transcription
initiation site. Sequences encoding metabolic pathway enzymes
provide particularly useful promoter sequences. Examples include
promoter sequences derived from sugar metabolizing enzymes, such as
galactose, lactose and maltose, and sequences derived from
biosynthetic enzymes such as tryptophan. Promoters from
bacteriophage may also be used and are known in the art. In
addition, synthetic promoters and hybrid promoters are also useful;
for example, the tac promoter is a hybrid of the trp and lac
promoter sequences. Furthermore, a bacterial promoter can include
naturally occurring promoters of non-bacterial origin that have the
ability to bind bacterial RNA polymerase and initiate
transcription.
[0094] In addition to a functioning promoter sequence, an efficient
ribosome binding site is desirable. In E. coli, the ribosome
binding site is called the Shine-Delgarno (SD) sequence and
includes an initiation codon and a sequence 3-9 nucleotides in
length located 3-11 nucleotides upstream of the initiation
codon.
[0095] The expression vector may also include a signal peptide
sequence that provides for secretion of the Toso protein in
bacteria. The signal sequence typically encodes a signal peptide
comprised of hydrophobic amino acids which direct the secretion of
the protein from the cell, as is well known in the art. The protein
is either secreted into the growth media (gram-positive bacteria)
or into the periplasmic space, located between the inner and outer
membrane of the cell (gram-negative bacteria).
[0096] The bacterial expression vector may also include a
selectable marker gene to allow for the selection of bacterial
strains that have been transformed. Suitable selection genes
include genes which render the bacteria resistant to drugs such as
ampicillin, chloramphenicol, ierythromycin, kanamycin, neomycin and
tetracycline. Selectable markers also include biosynthetic genes,
such as those in the histidine, tryptophan and leucine biosynthetic
pathways.
[0097] These components are assembled into expression vectors.
Expression vectors for bacteria are well known in the art, and
include vectors for Bacillus subtilis, E. coli, Streptococcus
cremoris, and Streptococcus lividans, among others.
[0098] The bacterial expression vectors are transformed into
bacterial host cells using techniques well known in the art, such
as calcium chloride treatment, electroporation, and others.
[0099] In one embodiment, Toso proteins are produced in insect
cells. Expression vectors for the transformation of insect cells,
and in particular, baculovirus-based expression vectors, are well
known in the art.
[0100] In a preferred embodiment, Toso protein is produced in yeast
cells. Yeast expression systems are well known in the art, and
include expression vectors for Saccharomyces cerevisiae, Candida
albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces
fragilis and K. lactis, Pichia guillerimondii and P. pastoris,
Schizosaccharomyces pombe, and Yarrowia lipolytica. Preferred
promoter sequences for expression in yeast include the inducible
GAL1,10 promoter, the promoters from alcohol dehydrogenase,
enolase, glucokinase, glucose-6-phosphate isomerase,
glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,
phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase,
and the acid phosphatase gene. Yeast selectable markers include
ADE2, HIS4, LEU2, TRP1, and ALG7, which confers resistance to
tunicamycin; the neomycin phosphotransferase gene, which confers
resistance to G418; and the CUP1 gene, which allows yeast to grow
in the presence of copper ions.
[0101] The Toso protein may also be made as a fusion protein, using
techniques well known in the art. Thus, for example, for the
creation of monoclonal antibodies, if the desired epitope is small,
the Toso protein may be fused to a carrier protein to form an
immunogen. Alternatively, the Toso protein may be made as a fusion
protein to increase expression, or for other reasons. For example,
when the Toso protein is a Toso peptide, the nucleic acid encoding
the peptide may be linked to other nucleic acid for expression
purposes.
[0102] In one embodiment, the Toso nucleic acids, proteins and
antibodies of the invention are labeled. By "labeled" herein is
meant that a compound has at least one element, isotope or chemical
compound attached to enable the detection of the compound. In
general, labels fall into three classes: a) isotopic labels, which
may be radioactive or heavy isotopes; b) immune labels, which may
be antibodies or antigens; and c) colored or fluorescent dyes. The
labels may be incorporated into the compound at any position.
[0103] In a preferred embodiment, the Toso protein is purified or
isolated after expression. Toso proteins may be isolated or
purified in a variety of ways known to those skilled in the art
depending on what other components are present in the sample.
Standard purification methods include electrophoretic, molecular,
immunological and chromatographic techniques, including ion
exchange, hydrophobic, affinity, and reverse-phase HPLC
chromatography, and chromatofocusing. For example, the Toso protein
may be purified using a standard anti-Toso antibody column.
Ultrafiltration and diafiltration techniques, in conjunction with
protein concentration, are also useful. For general guidance in
suitable purification techniques, see Scopes, R., Protein
Purification, Springer-Verlag, NY (1982). The degree of
purification necessary will vary depending on the use of the Toso
protein. In some instances no purification will be necessary.
[0104] Once expressed and purified if necessary, the Toso proteins
and nucleic acids are useful in a number of applications.
[0105] In a preferred embodiment, modified Toso cell-surface
receptors, and cells containing the modified receptors, are made.
In one embodiment, non-human animals (preferably transgenic) are
made that contain modified Toso receptors; similarly, "knock-out"
animal models and Toso transgenic animals that may contain an
inducible promoter may be made. In addition, cells, particularly
mammalian, can be made comprising a modified Toso cell surface
receptor.
[0106] In one embodiment, the Toso proteins of the present
invention may be used to generate polyclonal and monoclonal
antibodies to the full length protein, the extracellular or
cytoplasmic domains of Toso proteins, which are useful as described
herein. Similarly, the Toso proteins can be coupled, using standard
technology, to affinity chromatography columns. These columns may
then be used to purify Toso antibodies. In a preferred embodiment,
the antibodies are generated to epitopes unique to the Toso
protein; that is, the antibodies show little or no cross-reactivity
to other proteins. These antibodies find use in a number of
applications. For example, the Toso antibodies may be coupled to
standard affinity chromatography columns and used to purify Toso
proteins. The antibodies may also be used as described below.
[0107] In a preferred embodiment, antibodies, particularly
monoclonal antibodies, are used to modulate the biological function
of a Toso protein. "Modulating the activity of Toso" includes an
increase in activity, a decrease in activity, or a change in the
type or kind of activity present. By "Toso activity" or grammatical
equivalents herein is meant the ability of Toso after activation to
modulate apoptosis. As outlined herein, upon T cell activation,
Toso is activated, initiating a signalling pathway that results in
modulation of apoptosis. Such modulation may result in response to
either of the extracellular or cytoplasmic domains of Toso and may
correspond to a decrease or an increase in apoptosis. In a
preferred embodiment, the activity of Toso (either or both of the
extracellular or cytoplasmic domain of Toso) is increased; in
another preferred embodiment, the activity of Toso is
decreased.
[0108] In a preferred embodiment, methods of modulating apoptosis
in a cell are provided, comprising administering to the cell a
recombinant nucleic acid encoding a Toso protein, or a Toso
protein. This includes treating an apoptosis related condition or
apoptosis mediated disorders, which include, but are not limited
to, any disease characterized by T cell overactivity, including,
but not limited to Sjogrens mixed connective tissue disease,
autoimmune disorders including, but not limited to, lupus (SLE),
rheumatoid arthritis (RA), multiple sclerosis, and autoimmune
diseases which are tissue specific, for example liver (hepatitis),
kidney (nephritis) or Hashimoto (thyroiditis); diseases where T
cells actively destroy cells, for example, cytotoxic effects
including, but not limited to, transplant rejection, disease
conditions based on graft vs. host or host vs. graft reactions;
conditions where cells of any kind that are not dying express Toso
appropriately, for example, cancer of T or B cell origin (where
increased apoptosis would be desirable), including but not limited
to, leukemias and lymphomas, Chrohn's disease, skin inflammatory
disorders (psoriasis, eczema); and diseases secondary to altered
immunoglobulin production such as Waldenstroms, and multiple
myeloma.
[0109] In one embodiment, a therapeutically effective dose of a
Toso is administered to a patient. This may be done either by the
administration of a Toso protein, or a nucleic acid encoding a Toso
protein, as is known in the art. By "therapeutically effective
dose" herein is meant a dose that produces the effects for which it
is administered. The exact dose will depend on the purpose of the
treatment, and will be ascertainable by one skilled in the art
using known techniques. As is known in the art, adjustments for
Toso degradation, systemic versus localized delivery, and rate of
new protease synthesis, as well as the age, body weight, general
health, sex, diet, time of administration, drug interaction and the
severity of the condition may be necessary, and will be
ascertainable with routine experimentation by those skilled in the
art.
[0110] A "patient" for the purposes of the present invention
includes both humans and other animals, particularly mammals, and
organisms. Thus the methods are applicable to both human therapy
and veterinary applications. In the preferred embodiment the
patient is a mammal, and in the most preferred embodiment the
patient is human.
[0111] The administration of the Toso proteins of the present
invention can be done in a variety of ways, including, but not
limited to, orally, subcutaneously, intravenously, intranasally,
transdermally, intraperitoneally, intramuscularly, intrapulmonary,
vaginally, rectally, or intraocularly. In some instances, for
example, in the treatment of wounds and inflammation, the Toso may
be directly applied as a solution or spray.
[0112] The pharmaceutical compositions of the present invention
comprise a Toso protein in a form suitable for administration to a
patient. In the preferred embodiment, the pharmaceutical
compositions are in a water soluble form, such as being present as
pharmaceutically acceptable salts, which is meant to include both
acid and base addition salts. "Pharmaceutically acceptable acid
addition salt" refers to those salts that retain the biological
effectiveness of the free bases and that are not biologically or
otherwise undesirable, formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid and the like, and organic acids such as acetic
acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid and the like. "Pharmaceutically acceptable base
addition salts" include those derived from inorganic bases such as
sodium, potassium, lithium, ammonium, calcium, magnesium, iron,
zinc, copper, manganese, aluminum salts and the like. Particularly
preferred are the ammonium, potassium, sodium, calcium, and
magnesium salts. Salts derived from pharmaceutically acceptable
organic non-toxic bases include salts of primary, secondary, and
tertiary amines, substituted amines including naturally occurring
substituted amines, cyclic amines and basic ion exchange resins,
such as isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, and ethanolamine.
[0113] The pharmaceutical compositions may also include one or more
of the following: carrier proteins such as serum albumin; buffers;
fillers such as microcrystalline cellulose, lactose, corn and other
starches; binding agents; sweeteners and other flavoring agents;
coloring agents; and polyethylene glycol. Additives are well known
in the art, and are used in a variety of formulations.
[0114] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All references cited herein
are incorporated by reference.
EXAMPLE I
[0115] Molecular Cloning and Chromosomal Localization of Toso.
[0116] Jurkat cells (human T cell line, a gift of Dr. Calvin Kuo,
Harvard Univ.) were infected with a retroviral Jurkat T cell cDNA
library to screen for cDNAs that encode inhibitory molecules for
Fas-induced apoptosis. A retroviral library containing
2.times.10.sup.6 independent cDNA inserts was constructed from
Jurkat cell mRNA by standard methods (Kinoshita and Nolan,
unpublished) using a retrovirus vector pBabeMN (Kinoshita, et al.
(1997)). The library was transfected into an ecotropic virus
packaging cell line, .phi.NX-Ampho, as described previously. Jurkat
cells were spin-infected with the supernatant from .phi.NX-A cells
resulting in 20-40% infection using this method as determined by
doping of the library with a marker retrovirus pBabeMN-LacZ or
pBabeMN-Lyt-2-.alpha. (194 amino acids), which does not have
cytoplasmic domain (Tagawa, et al., Proc. Natl. Acad. Sci,
83:3422-3426 (1986)). Jurkat cells were aliquoted into 96-well
plates in media containing 10 ng/ml of anti-human Fas mAb, CH11,
(Kamiya Biomedical Company, CA 91359, U.S.A.) for 15 days. Jurkat
cells, under conditions empirically derived, were sensitive to
Fas-mediated apoptosis with a spontaneous survival rate under our
conditions of 2-3 per 10.sup.6 cells. Cells that survived the
Fas-mediated killing were identified by outgrowth in the 96 well
plate format, expanded, total RNA extracted, and cDNA inserts
rescued using RT-PCR (AMV reverse transcriptase from Promega, Wiss.
53711, U.S.A. and Vent DNA polymerase from New England Biolabs,
Inc., Mass. 01915, U.S.A.) with primers 5'-GCT CAC TTA CAG GCT CTC
TA (LibS) and 5'-CAG GTG GGG TCT TTC ATT CC (LibA), which were
located 282 bp and 56 bp nucleotides upstream and downstream of
cDNA insert cloning sites. After an initial denaturation at
94.degree. C. for 5 minutes, each cycle of amplification consisted
of 30 second denaturation at 94.degree. C., followed by a 30
second-annealing at 58.degree. C. and 2 minutes extension at
72.degree. C. After 35 cycles, the final product was extended for
10 minutes at 72.degree. C. The rescued inserts were digested with
BamHI-Sall (Promega) or BstXI (Promega), and ligated into the
pBabeMN retrovirus vector. The cloned retrovirus containing the
novel insert was infected into Jurkat cells. Cells were cultured
with 10 ng/ml anti-Fas mAb to confirm whether the inhibitory effect
was caused by cDNA inserts of retrovirus. 26 clones were obtained
that were resistant to Fas-induced apoptosis, of which 12 carried
cDNA inserts. After a second round of anti-Fas screening, one
clone, termed here Toso, demonstrated potent inhibition of
Fas-induced apoptotic signaling.
[0117] The cDNA insert of Toso was found to contain a 5'-non-coding
region of 73 nucleotides, a coding region of 1173 nucleotides (390
amino acids) and a 3'-non-coding region of 665 nucleotides. (See
FIG. 1, SEQ ID NO:1). The ATG initiation codon is contained within
a standard Kozak consensus sequence. Kyte-Doolittle hydropathy plot
analysis showed that Toso has two hydrophobic regions: the
amino-terminal residues from 1 to 17 correspond to the deduced
signal sequence (underlined) and residues from 254 to 272 (double
underlined) correspond to a presumptive transmembrane region
[Hofmann and Stoffel, f993, analysis was performed using DNAsis-Mac
V2.0 (Hitachi Software Engineering, Co. Ltd., Japan)], suggesting
that Toso is a type I integral membrane protein. (See FIG. 2b). The
predicted molecular weight of Toso is 41 kDa. The cytoplasmic
region of Toso has a basic amino acid-rich region (from R.sup.274
to R.sup.323), a proline-rich region (from P.sup.334 to P.sup.346),
and an acidic amino acid-rich region (from E.sup.378 to D.sup.3")
(See FIGS. 2a and 2b, SEQ ID NO: 2). BLAST search analysis revealed
that Toso is a unique gene (Altschul, et al., (1990)). The
extracellular domain of Toso has homology to the immunoglobulin
variable (IgV) domains, which is characterized by motifs in the
.beta.-strand B, D and F regions, (residues VTLTC, RV (or F, 1) and
DSG (or A)-Y-CA) (Williams and Barclay, Ann. Rev. Immunol.,
6:381-405 (1988)). Importantly, the cysteines in the IgV-like motif
VTIKC at position 33 in Toso, as well as the cysteine in the
IgV-like motif DSGVYAC at position 98, are appropriately distanced
as in other IgV-like domains to form a disulphide bond. Toso also
contains within the Ig domains two additional cysteines that are
not conserved in other IgV-like domains. Thus, the presumptive
extracellular domain has all the requisite features that demarcate
it as a potential IgV-like domain. The cytoplasmic region of Toso
has partial homology to FAST kinase, acid sphingomyelinase, insulin
receptor substrate-1 (IRS-1) and the apoptosis inhibitor from
Orgyia pseudotsugata nuclear polyhedrosis virus (Op-1AP) (FIG. 3),
which might function to initiate some of the signaling systems
acted upon by Toso.
[0118] The Toso gene was mapped to a human chromosome by using a
panel of 17 human X Chinese hamster hybrid cell lines derived from
several independent fusion experiments (Francke et al., 1986). PCR
primers used to amplify Toso sequence derived from the 3'
untranslated region were 5'-AGA GGC ATA GCT ATT GTC TCG G (sense;
located 369 bp downstream of the coding region), and 5'-ACA TTT GGA
TCA GGG CAA AG (anti-sense; 508 bp downstream of the coding
region). The size of the PCR product was 159 bp. The PCR conditions
were 94.degree. C., 90 seconds; then 35 cycles of 94.degree. C., 20
seconds; 55.degree. C., 30 seconds; 72.degree. C., 45 seconds;
followed by 72.degree. C., 5 minutes. Specific PCR products were
obtained from human genomic DNA, and hybrid cell lines that carry
human chromosome 1. The PCR product was sequenced to confirm its
identity.
[0119] To map the Toso gene locus more precisely, two human
radiation hybrid (RH) mapping panels were typed by PCR. GeneBridge
4 (Whitehead Institute/MIT Genome Center) and Stanford G3 (Stanford
Human Genome Center), were obtained from Research Genetics, Inc.
(Cox, et al., Science, 250:245-250 (1990); Walter, et al., Net
Genet, 7:22-28 (1994)), and samples were typed using the primers
and PCR conditions described above. Results of the maximum
likelihood analysis (Boehnke, et al., Am. J. Hum. Genet.,
49:1174-1188 (1991)) were obtained by submitting the raw scores to:
http://www-genome.wi.mit.edu/cgibin/contig/rhmapper.pl and
http://wwwshgc.stanford.edu/rhserver2/rhserver_form.html. The
cytological localization of the Toso gene was deduced from the
cytogenetic information about the flanking markers in Bray-Ward et
al (Bray-Ward, et al., Genomics, 32:1-14 (1996)). In the Stanford
G3 mapping panel, Toso cosegregated with chromosome 1 marker
D1S3553 on all 83 Stanford G3 panel RH cell lines. D1S3553 is a
known marker of chromosome 1 bin 115 on the SHGC RH map. In the
GeneBridge 4 mapping panel, Toso is located 5.4 cR.sub.3000 and 1.7
cR.sub.3000 from D1 S504 and W1-9641, respectively. The order of
loci in this region from centromere to qter is: D1S412- D1S306
D1S504-Toso- W1-9641-D1S491- D1S237. According to Bray-Ward et al.
(1996), the YACs containing the more proximal markers D1S412 (bin
104), D1S477 (bin 109) and D1S504 (bin 114) were mapped to
1q25-q32, 1q31-q32 and 1q25-q32 respectively, and the YACs
containing the more distal markers D1S491 (bin 118), D1S414 (bin
121) and D1S237 (bin 124) were mapped to essentially the same
region, 1q31-q32, q31-q32 and 1q32-q41, respectively. Thus, the
Toso gene is located at 1q31-q32, a region in which several
chromosomal abnormalities relating to leukemias are localized.
[0120] Toso is a negative regulator of Fas-mediated cell death in
lymphoid cells, and may therefore be involved in oncogenic events
or resistance to chemotherapy (Friesen, et al., Nature Medicine,
2:574-577 (1996)). The gene for Toso localizes within human
chromosome region 1q31-q32. Chromosomal changes in 1q32 are
frequently observed in human cancer, including various types of
hematopoietic malignancies and solid tumors (Jinnai, et al., Am. J.
Hematol, 35:118-124 (1990); Mertens, et al., Cancer Res.,
57:2765-2780 (1997); Mitelman, et al., Nat. Genet., 417-474 (1997);
Schrnid and Kohler, Cancer Genet. Cytogenet, 11: 121-23 (1984);
Shah, et al., Cancer Genet. Cytogenet, 61:183-192 (1992); Waghray,
et al., Cancer Genet. Cytogenet, 23:225-237 (1986); Yip, et al.,
Cancer Genet. Cytogenet, 51:235-238 (1991)). Furthermore, studies
in nude mice demonstrated that duplication of the chromosome
segment of 1 q11-q32 is associated with proliferation and
metastasis of human chronic lymphocytic leukemic B-cells (Ghose, et
al., Cancer Res., 50:3737-3742 (1990)), suggesting the presence of
dominantly acting growth regulatory or cell survival genes. Thus,
Toso is a candidate for evaluation as a proto-oncogene in several
proliferative and metastatic neoplasms.
EXAMPLE 2
[0121] Toso Inhibits Fas-, TNF.alpha.- and FADD-Induced
Apoptosis.
[0122] Jurkat cells that express the receptor for ecotropic murine
retroviruses ("Jurkat.ecoR") were infected with retroviruses that
express Toso and control vectors, pBabeMN-Toso, pBabeMN-lacZ and
pBabeMN-Lyt-2-.alpha.' (.alpha.' form of mouse CD8.alpha. chain)
(Tagawa, et al. (1986)). Cells were cultured in the presence of
several reagents such as anti-Fas mAb (Kamiya Biomedical Company),
staurosporine (SIGMA Chemical Company, MO 63178, U.S.A.), ceramide
(SIGMA), PMA (SIGMA)/lonomycin (SIGMA), human TNF-.alpha. (R&D
systems, Minneapolis, Min. 55413)/Cycloheximide (SIGMA). After 12
or 24 hours, the cells were stained with 100 .mu.g/ml ethidium
bromide (SIGMA) and 100 .mu.g/ml acridine orange (SIGMA). Apoptotic
cells and non-apoptotic cells were identified with UV microscopy as
described (MacGahon, et al., The End of the (Cell)Line: Methods for
the Study of Apoptosis in vitro, in Methods in cell biology, L. J.
Schwartz and B. A. Osborne, eds., San Diego, Calif., Academic
Press, Inc., pp. 172-173 (1995)). For FADD-induced apoptosis, mouse
FADD (a gift from Dr. Angeles Estelles, Dept. Mol. Pharm., Stanford
Univ.) was ligated into pBabeMN retroviral vector. Jurkat.ecoR
cells expressing Lyt-2-.alpha.' or Toso were infected with
pBabeMN-LacZ or pBabeMN-FADD. After 24 hours infection with FADD,
the cells were stained with ethidium bromide and acridine orange
and counted the apoptotic cells. Jurkat.ecoR cells were infected
with pBabeMN-lacZ, pBabeMN-Lyt-2-.alpha.', and pBabeMN-Toso. At 72
hours postinfection, infection frequency of pBabeMN-lacZ and
pBabeMN-Lyt-2.alpha.' were determined to be 45% and 58%,
respectively. Jurkat cells were then cultured with 10 ng/ml
anti-Fas mAb for 24 hours and apoptotic cells were counted.
Jurkat.ecoR cells expressing Toso were resistant to apoptosis
induced by 10 ng/ml of anti-Fas mAb, whereas Jurkat cells,
Jurkat.ecoR cells and Jurkat.ecoR cells that expressed lacZ or
Lyt-2-.alpha.', all succumbed to apoptotic death (FIG. 4a).
[0123] Staurosporine is a bacterial alkaloid that is a broad
spectrum inhibitor of protein kineses (Tamaoki and Nakano,
Biotechnology, 8:732-735 (1990)) and induces programmed cell death
in various cell lines and dissociated primary cells in culture
(Ishizaki, et al., J. Cell Biol., 121:899-908 (1993); Jacobson, et
al., Nature, 361:365-369 (1993); Raff, et al., Science, 262:695-700
(1993)). Ceramide generation is implicated in a signal transduction
pathway that mediates programmed cell death induced by Fas and
TNF-.alpha. (Cifone, et al., J. Exp. Med., 180:1547-1552 (1994);
Obeid, et al., Science, 259:1769-1771 (1993)). pBabeMN-LacZ
infected cells were counted by microscopic observation; infection
frequency was determined to be 57%. At 72 hours postinfection,
Jurkat.ecoR cells and Jurkat.ecoR cells infected with pBabeMN-lacZ
and pBabeMN-Toso were cultured with anti-Fas mAb, staurosporine and
ceramide for 24 hours. Although Jurkat.ecoR cells expressing Toso
were resistant to Fas-mediated apoptosis over a range of antiFas
dilutions, these cells were not resistant to any concentration of
staurosporine- or ceramide-induced apoptosis (FIG. 4b).
[0124] The Fas receptor has homology to the TNF-A receptor, and
these two receptors share analogous signaling systems as well as
several intracellular mediators (Hsu, et al., Cell, 84:299-308
(1996)). The protective effect of Toso against TNF-.alpha.-induced
apoptosis was tested by culturing Jurkat.ecoR cells expressing
Lyt-2-.alpha.' or Toso with 10 ng/ml of anti-Fas mAb or 1 .mu.g/ml
of TNF-.alpha. in the presence of 0.1 .mu.g/ml of cyclohexamide
(CHX) for 12 hours and apoptotic cells were counted. The infection
frequency of pBabeMN-Lyt-2-.alpha.' was determined to be 58%. Toso
inhibited Fas induced apoptosis in the presence of CHX and also
protected against TNF-.alpha.-induced apoptosis in comparison to
Jurkat.ecoR expressing Lyt-2-.alpha.' (FIG. 4d). Thus the
TNF-.alpha. and Fas signaling pathways may converge at a common
point that can be inhibited by Toso.
[0125] Fas-Mediated Apoptosis is Activated through FADD.
[0126] The effect of Toso on FADD-induced apoptosis was
investigated by infecting Jurkat.ecoR cells expressing
Lyt-2-.alpha.' or Toso, with pBabeMN-LacZ or pBabeMN-FADD. The
reinfection efficiency was approximately 40% using pBabeMN-LacZ.
Jurkat.ecoR cells were infected with pBabeMN-Lyt-2-.alpha.', and
pBabeMN-Toso. Infection frequency of pBabeMN-Lyt-2-.alpha.' was
determined to be 72%. Jurkat.ecoR cells expressing Lyt-2-.alpha.'
or Toso were infected with pBabeMNLacZ or pBabeMN-FADD and
apoptotic cells were counted at 24 hourrs postinfection. Infection
frequency of pBabeMN-lacZ in Jurkat.ecoR cells expressing
Lyt-2-.alpha.' and Toso was determined to be 39% and 43%,
respectively. As shown in FIG. 4c, FADD induced apoptosis in 45% of
control Jurkat cells. However, FADD failed to induce apoptosis in
Jurkat.ecoR cells constitutively expressing Toso.
[0127] The downstream effects of Toso on known inhibitors of
apoptosis, were evaluated by western blot analysis of Bc1-2 and BCI
XL expression levels in Toso expressing cells. For detection of
Toso or deletion mutants that has a HA tag, whole-cell lysates
(2.times.10.sup.5 cells per lane) were resolved by SDS-PAGE,
transferred to an Immobilon-P transfer membrane (Millipore,
Bedford, Mass. 01730, U.S.A.) and processed using ECL western
blotting analysis system (Amersham Life Science, Arlington Heights,
Ill. 60005, U.S.A.) with Mouse monoclonal anti-hemagglutinin
antibody (HA. 11) (Babco, Richmond, Calif. 94804, U.S.A.) as per
manufacturer recommendation. Bc1-2 overexpression can block
Fas-induced apoptosis as well as staurosporine-induced apoptosis
(data not shown). No change in the levels of expression of Bc1-2 or
BCI XL was observed by Western blot (data not shown). Thus, it
appears that intracellular signaling events generated by FADD can
be directly and efficiently blocked by signals emanating from Toso
at a point prior to engagement of Bc1-2 and BCI XL. The results
also suggest that Toso's effect is not due to down regulation of
FADD gene expression.
[0128] The effect of overexpression of Toso on processing of
caspase-8, which associates with FADD, was evaluated. The processed
form (p20) of FLICK after Fas activation was greatly reduced in
pBabeMN-Toso-infected Jurkat.ecoR cells in comparison with control
Jurkat.ecoR cells (see FIG. 5a). To detect caspase-8, whole-cell
lysates (2.times.10.sup.6 cells per lane) were resolved by
SDS-PAGE, transferred to an membrane and processed with goat
anti-Mch5 p20 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz,
Calif. 95060, U.S.A.) as described above. This data indicates that
Toso inhibits caspase-8 processing after Fas activation. cFLIP
expression was induced by Toso (FIG. 5b). These results strongly
suggest that the extracellular domain of Toso inhibits Fas-induced
apoptosis by preventing caspase-8 processing through cFLIP
upregulation. Toso did not inhibit staurosporine-induced programmed
cell death and staurosporine has been shown to activate caspase-8
(Jacobsen, et al., J. Cell Biol., 133:1041-1051 (1996)). Therefore,
additional Toso effects do not occur downstream, nor at the level,
of caspase-8. Supporting this, Toso also did not inhibit
ceramide-induced apoptosis, which acts downstream or independent of
caspase-8 as demonstrated in experiments using the
caspase-8-specific inhibitor peptide, DEVD-CHO (Gamen, et al., FEBS
Lett., 390:232-237 (1996)), which does not inhibit ceramide-induced
apoptosis. Overexpression of Bc1-2 or Bc1-XL is known to prevent
apoptosis in response to ceramide and staurosporine (Geley, et al.,
FEBS Lett., 400:15-18 (1997); Susin, et al., J. Exp. Med.,
186:25-37 (1997); Takayama, etal., Cell, 80:279-284 (1995); Zhang,
etal., Proc. Natl. Acad. Sci. USA, 93:5325-5328 (1996)). Toso did
not change the expression levels of Bc1-2 nor BCL-XL in Jurkat
cells, showing that neither Bc1-2 nor BCL-XL were involved in the
protective activities of Toso. Taken together then, Toso activates
an inhibitory pathway that prevents caspase-8 activation following
Fas stimulation through upregulation of cFLIP, and not by blocking
apoptotic signals downstream or at the level of caspase-8. This
explains the apparent specificity of the blockade to TNF
family-related surface receptors that use caspase-8 for apoptotic
signaling.
[0129] Cells expressing Toso alone were mixed with an equal number
of cells expressing lacZ. After one round of Fas stimulation, no
lacZ-expressing cells remained as assayed by X-gal. In addition,
Jurkat.ecoR cells were infected with pBabeMN-Toso-IRES-GFP. After
infection, cells were cultured with (.alpha.-Fas (+)) or without
(.alpha.-Fas (-)) 50 ng/ml of anti-Fas mAb. In the absence of
anti-Fas mAb treatment (Fas (-))., 46% GFP negative cells and 54%
GFP positive cells were observed in pBabeMN-Toso-IRES-GFP-infected
Jurkat.ecoR cells. After five days culture with anti-Fas mAb,
survivors were obtained from pBabeMN-Toso-IRES-GFP-infected
Jurkat.ecoR cells, but not from control pBabeMN-IRES-GFP-infected
Jurkat.ecoR cells (data not shown); 99.7% of surviving Jurkat cells
expressed GFP as shown in FIG. 4e (Fas(+)). These data indicate
that cells that express the extracellular domain of Toso are
protected from Fas-induced apoptosis and suggests that Toso does
not exert its effect as a secreted form.
EXAMPLE 3
[0130] The Immunoglobulin Domain and the Transmembrane Region of
Toso Are Required for Inhibition of Fas-Induced Apoptosis.
[0131] The C-terminus deletion mutants (Toso.DELTA.(377-390).HA,
Toso.DELTA.(334-390).HA, Toso.DELTA.(281-390).HA and
Toso.DELTA.(252-390).HA), the N-terminus deletion mutant
(Toso.DELTA.(29-187).HA) and the fusion protein
(Lyt-2/Toso(271-390).HA) of the extracellular domain and
transmembrane region from Lyt-2-.alpha.' and the cytoplasmic domain
from Toso, which have the influenza virus hemmagglutinin tag (HA)
in C-terminus, were generated by. Primers in the antisense
orientation, carrying the 20 nucleotide sequences of Toso located
upstream of the deletion sites, HA tag sequence and an in-frame
termination codon, as well as Ncol site, were synthesized. The DNA
fragment of the Toso gene from the Xhol site located in the
extracellular domain to the Ncol site that is located in 3'
non-coding region was replaced with the PCR products amplified from
pBabeMN-Toso using LibS and each primer described above. A primer
for Toso.DELTA.(29-187).HA in the antisense orientation carrying
the 20 nucleotides located after the leader peptides of Toso and
Xhol site was synthesized. The DNA fragment from Dralil site, which
is located 190 bp upstream of cDNA insert cloning sites, to Xhol
site in pBabeMN-Toso.HA was replaced with the PCR product amplified
from pBabeMN-Toso using LibS and the primer. For
Lyt2/Toso(271-390).HA, primer in sense orientation which is carried
a BamHI site and the 20 nucleotides located upstream of the
cytoplasmic domain was synthesized. The DNA fragment from Bc1 I
site, which is located in the end of transmembrane region of
Lyt-2-.alpha.', to Sall site, which is located downstream of
Lyt-2-.alpha.' cloning sites in pBabeMN-Lyt-2-.alpha.', was
replaced with the PCR product amplified from pBabeMN-Toso.HA using
LibA and the primer. All mutants generated by PCR were verified by
DNA sequencing using cycle sequencing ready reaction kit. Toso
deletion mutants prepared as described above were epitope-tagged in
order to delineate the regions responsible for anti-apoptotic
signal transduction, (FIGS. 6a and 6b). Toso.HA (fused to the
hemagglutinin, HA, tag) had an apparent molecular weight of 60 kDa,
suggesting Toso is heavily glycosylated. The cell surface
expression of Fas using anti-human Fas mAb, CH 11, was determined
by FACS to explore whether Toso has an effect on Fas expression.
Fas was expressed at similar levels on the surface of cells
expressing either full-length Toso, Toso deletion mutants, or
control vector. Thus, the extracellular domain of Toso neither
downregulates Fas, nor directly interferes with the ability of the
antibody to bind and presumably stimulate Fas.
[0132] Jurkat.ecoR cells were infected with pBabeMN-Lyt-2-.alpha.'.
HA, pBabeMN-Toso. HA, pBabeMN-Toso.DELTA.(377-390). HA,
pBabeMN-Toso.DELTA.(334-390). HA, pBabeMN-Toso.DELTA.(281-390).HA,
pBabeMN-Toso.DELTA.(252-390).HA and pBabeMN-Toso.DELTA.
(29-187).HA. Jurkat cells were cultured with 10 ng/ml anti-Fas mAb
for 24 hours and apoptotic cells were counted. Apoptosis was
readily induced in control Jurkat.ecoR cells and Jurkat.ecoR cells
expressing Lyt-2-.alpha.'.HA, whereas apoptosis was markedly
inhibited in Jurkat.ecoR cells that expressed Toso.HA (FIG. 6a).
Deletions of regions of the cytoplasmic domain of Toso from 334 to
390 still inhibited apoptosis. Moreover, a deletion of Toso lacking
the entire cytoplasmic domain still retained substantial
anti-apoptotic ability. Thus, the cytoplasmic domain of Toso is not
absolutely required for the anti-apoptotic effects on Fas
antibody-stimulated cells. (See Example 5, below) These results
indicate that the homologies observed in the cytoplasmic region of
Toso, as shown in FIG. 3, are not the only sources of the
anti-apoptotic signals generated by a Toso complex, although the
cytoplasmic regions are required for enhancing the anti-apoptotic
effects of Toso.
[0133] The Toso mutant lacking the transmembrane and cytoplasmic
domains demonstrated that inhibition of Fas-induced apoptosis by
Toso requires its insertion into membranes. As shown in FIG. 6a,
soluble Toso.DELTA.(252-390).HA afforded no protection from
apoptosis. Expression of the Toso.DELTA.(252-390).HA protein was
confirmed by western blot analysis of culture supernatants.
Supernatants derived from pBabeMN-Toso.DELTA.(252-390).
HA-transfected 293T cells did not inhibit Fas-induced apoptosis,
indicating that a membrane-proximal event dependent on
cis--localization of Toso is required for blockade of the
Fas-mediated death signal.
[0134] Many cell surface receptor complexes act through
oligomerization and most immunoglobulin (Ig) domain proteins exist
in homodimeric and heterodimeric Ig forms, functioning as
self-assembling systems. Disruption of the Ig domain of Toso
completely abrogated the anti-apoptotic ability of Toso
(Toso.DELTA.(29-187).HA). (See FIG. 6a). Further, a chimeric
Lyt-2Toso fusion protein in which the cytoplasmic domain of Toso
was coupled to the extracellular and transmembrane region of
Lyt-2-.alpha.' (.alpha.' form of murine CD8.alpha., which forms
homodimers at the cell surface) (Tagawa, et al., (1986)) failed to
inhibit Fas-induced apoptosis. Furthermore, anti-mouse CD8a mAb
(Lyt-2) was used to crosslink the Lyt-2-Toso chimeras and induce
multimerization of the Toso cytoplasmic domains. Toso.HA-expressing
Jurkat.ecoR cells (5.times.10.sup.6 cells) were incubated with 2 mM
BS3 (PIERCE, Rockford, Ill. 61105, U.S.A.) for 1 hour at 4.degree.
C. After incubation, 1 M Tris-HCI was added to a final
concentration of 10 mM and cells were incubated for 15 minutes at
4.degree. C. Whole-cell lysates were resolved by SDS PAGE,
transferred to a membrane and processed with mouse monoclonal
antihemagglutinin antibody (HA.11) (Babco) as described above.
Jurkat.ecoR cells and Toso-expressing Jurkat.ecoR cells were used
as controls. Jurkat.ecoR cells which expressed the chimeric
Lyt-2-Toso fusion protein did not show any protection against
anti-Fas mAb-induced apoptosis in presence of anti-mouse CD8.alpha.
mAb. These results suggest that some form of Ig domain mediated
dimerization of Toso is required to initiate the anti-apoptotic
effect in conjunction with the cytoplasmic region of Toso or other
cell surface Toso-associating proteins. Cell surface molecules on
Toso.HAexpressing Jurkat.ecoR cells were crosslinked using the
water-soluble crosslinker, BS3 and apparent crosslinking molecular
complexes at 150, 240, 300 kDa were detected (See FIG. 6c). This
result first indicates that Toso is a surface expressed receptor.
The results are consistent with an association of Toso with another
surface protein(s) of molecular weight 90 kDa. The several
molecular weights observed for the crosslinked complexes are also
minimally consistent with stochiometric mixtures of 60 and 90 kDa
molecules.
[0135] Domain Analysis of Toso Suggests Multiple Interacting
Partners
[0136] Deletion analysis of Toso indicated that surface expression
of the immunoglobulin V-like region is necessary to inhibit
Fas-induced-apoptosis and that the cytoplasmic domain of Toso is
insufficient and indeed partly expendable for the anti-apoptotic
function. Deletion of the cytoplasmic domain resulted in abrogation
of only about half of the anti-apoptotic effect. This suggests that
Toso must be expressed at the cell surface in a manner where it
presumably interacts other surface molecule(s) that propagate an
anti-apoptotic signal. Most immunoglobulin family receptors are
homo- or heterodimers that can become activated through ligand
interactions. Crosslinking experiments revealed multiple potential
higher-order complexes (150, 240, and 300 kDa), suggesting at least
one partner of 90 kDa that interact with Toso. We suspect that Toso
forms a heterodimer with this other surface protein to collaborate
in initiating the anti-apoptotic signal that leads to cFLIP
induction. Interactions of surface-expressed Toso complexes with
ligands on or near target cells might also modulate the ability of
Toso to provide anti-apoptotic signaling. We are currently
investigating the existence of such ligands and contributory
molecules.
[0137] A model summarizing the results is shown in FIG. 10. In this
model, stimulation through of the T cell receptor complex transmits
activation signals leading to upregulation of Fas and FasL.
Activation also induces Toso expression, providing the potential
for anti-apoptotic signals that protect against Fas-mediated
apoptosis. Toso accomplishes this by forming homo- or heterodimers
at the cell surface to generate signals that inhibit the initiation
or propagation of caspase-8 activation by cFLIP. It is also
possible that Toso requires an extracellular ligand that might
modulate its activities. The signaling pathway activated by Toso is
clearly important as it leads to induced expression of cFLIP
(Irmler, et al. (1997); Srinivasula, et al. (1997)).
EXAMPLE 4
[0138] T Cell Signaling Leading to Apoptosis is Blocked by
Activated Toso.
[0139] Poly (A).sup.+ RNA was prepared from Jurkat cells or Jurkat
cells stimulated for 24 hours with 10 ng/ml of phorbol 12-myristate
13-acetate (PMA; SIGMA Chemical Company, Mo. 63178, U.S.A.) and 1
.mu.g/ml phytohemagglutinin (PHA; SIGMA) or 10 ng/ml PMA and 500
ng/ml lonomycin (SIGMA). Poly (A).sup.+ RNA (5.mu.g) was subjected
to electrophoresis through 1% agarose gel containing 2.2 M
formaldehyde, and transferred to Hybond N.sup.+ membrane (Amersham
Life Science Inc., Ill. 60005, U.S.A.). Hybridization was carried
out according to the manufacturer's recommendation. A specific
probe for the Toso coding region (1.2 kbp) was synthesized with PCR
from pBabeMN-Toso using 5'-AGG GGC TCT TGG ATG GAC (TosoS) and
5'-CTG GGG TTG GGG ATA GC (Toso.DELTA.). As a control probe, the
human .beta.-actin cDNA control probe (CLONTECH Laboratories, Inc.,
CA 94303-4230) was used. Probes were labeled with .sup.32P using a
random-primed labeling kit, Prime-a-Gene (Promega). Human RNA
Master Blot and Human Immune System Multiple Tissue Northern Blot
II (CLONTECH Laboratories) were used to survey Toso mRNA expression
in several human tissues. Toso expression was observed in lymph
nodes, lung and kidney. In addition to these tissues, we detected
faint signals from spleen, thymus, liver, heart and salivary gland.
Tissues which were analyzed for Toso mRNA include: A1: Whole brain,
A2: Amygdala, A3: Caudate nucleus, A4: Cerebellum, A5: Cerebral
cortex, A6: Frontal lobe, A7: Hippocampus, A8: Medulla oblongata,
B1: Occipital lobe, B2: Putamen, B3: Substantia nigra, B4: Temporal
lobe, B5: Thalamus, B6: Subthalamic nucleus, B7: Spinal cord, C1:
Heart, C2: Aorta, C3: Skeletal muscle, C4: Colon, C5: Bladder, C6:
Uterus, C7: Prostate, C8: Stomach, D1: Testis, D2: Ovary, D3:
Pancreas, D4: Pituitary gland, D5: Adrenal gland, D6: Thyroid
gland, D7: Salivary gland, D8: Mammary gland, E1: Kidney, E2:
Liver, E3: Small intestine, E4: Spleen, E5: Thymus, E6: Peripheral
leukocyte, E7: Lymph node, E8: Bone marrow, F1: Appendix, F2: Lung,
F3: Trachea, F4: Placenta, G1: Fetal brain, G2: Fetal heart, G3:
Fetal kidney, G4: Fetal liver, G5: Fetal spleen, G6: Fetal thymus,
G7: Fetal lung. (FIG. 7a). Using Human Immune System Multiple
Tissue Northern Blot II, and film exposed at -70.degree. C. with an
intensifying screen for one day, endogenous Toso mRNA species of
2.0 (major), 2.8, 3.5 and 4.3 kbp were detected in lymph node and
spleen (see FIG. 7b). The nucleotide length of the cDNA was 1.9
kbp, suggesting that the additional bands might either be
alternative splice products or incompletely processed messages.
Toso expression was also observed in peripheral blood leukocytes,
thymus (FIG. 7b). Expression in bone marrow and fetal liver was
much lower than that in lymph node and spleen, as seen after
overexposure of the blot (data not shown).
[0140] The expression of Toso in several human cell lines was
analyzed by semi-quantitative RT-PCR involving amplification of the
1.2 kbp-coding region of Toso (FIG. 7c). The first strand of cDNA
was synthesized with 10 .mu.g total RNA from several human cell
lines and peripheral blood mononuclear cells. PCR was performed for
35 cycles using TosoS and Toso.DELTA.. After an initial
denaturation at 94.degree. C. for 5 minutes, each cycle of
amplification consisted of 30 second denaturation at 94.degree. C.,
followed by a 30 second-annealing at 58.degree. C. and 2 minutes
extension at 72.degree. C. After 35 cycles, the final product was
extended for 10 minutes at 72.degree. C. PCR products were
electrophoresed through 1.0% agarose gel and transferred to Hybond
N+membrane. The BamHI-Xhol fragment (510 bp) of the Toso-coding
region were labeled with .sup.32p. Hybridization was carried out as
described above. To detect cFLIP mRNA expression, a 1.1 kbp
fragment (998-2061) of the cFLIP gene (U97074) was amplified with
primers 5'-GGG AGA AGT AAA GAA CAA AG and 5'-CGT AGG CAC AAT CAC
AGC AT for 35 cycles as described above. The sequence of the 1.1
kbp PCR product was verified using cycle sequencing ready reaction
kit (Perkin Elmer, Calif. 94404, U.S.A.). As a control, .beta.actin
cDNA was amplified for 15 and 25 cycles as described above.
[0141] Toso mRNA was detected in lymphoid cell lines such as Jurkat
cells (T cell leukemia), a kind gift from Dr. Kuo, Harvard Univ.,
CemT4 cells (T cell leukemia), MolT-4 cells (T cell leukemia),
HB11.19 cells (B cell Iymphoma), a kind gift from Dr. Cleary, M.
L., Stanford Univ., and Reh cells (acute lymphocytic leukemia; non
T; non B, ATCC). HL-60 cells (promyelocytic leukemia, ATCC)
displayed a consistently weak signal. In contrast, Toso PCR
products were not detected in non-hematopoietic cell lines
including HepG2 cells (hepatoblastoma, a kind gift from Dr. Blau,
Stanford Univ.), 293 cells (kidney; transformed with adenovirus,
ATCC) and Hela cells (cervix; adenocarcinoma, ATCC). Toso therefore
is constitutively expressed in cells of hematopoietic cells.
[0142] Poly (A).sup.+ RNA was prepared from Jurkat cells stimulated
for 24 hours with 10 ng/ml PMA (SIGMA) and 500 ng/ml lonomycin
(SIGMA). The first strand of cDNA was synthesized with 10 .mu.g
Poly (A).sup.+ RNA using oligo-dT primers and performed PCR with
primers, 5'-AGA ATT CTC TCT AGG GGC TCT TGG ATG (See FIG. 1 where
the EcoRI site is underlined) and 5'-ATA AAG CTT CTC AGG GCA CAG
ATA GAT GG (HindIII site is underlined), which were located 23 bp
and 136 bp nucleotides upstream and downstream of the Toso coding
region, respectively. The 1.3 kbp fragment was ligated into
pBluescript SK(+). Five independent clones were picked up and
sequenced using cycle sequencing ready reaction kit (Perkin Elmer).
The deduced amino-acid sequences from the five independent clones
were completely identical to the gene from the cDNA library
screening, although two silent mutations were found within the
original gene as compared to the PCR consensus sequences.
[0143] Toso was expressed in several human cell lines including
Jurkat cells, CemT4 cells (human T cell leukemia), SupT1 cells
(human T cell leukemia, a kind gift from Dr. Cleary, M. L.,
Stanford Univ.), Oli-Ly8 cells (human B cell line; transformed with
EBV), AMK cells (human B cell line; transformed with EBV), both a
gift from Dr. Negrin, R. S., Stanford Univ., Reh cells (acute
lymphocytic leukemia; non T; non B), HL-60 cells (promyelocytic
leukemia) and HepG2 cells (hepatoma) using pBabeMN-Toso IRKS neo to
allow cotranslational selection with Geneticin (GIBCO BRL). All of
the human T cell lines and one of the human B cell lines, Oil-Ly8
cells, in which Toso was overexpressed, were inhibited for
apoptosis induced by anti-Fas mAb, whereas no significant
protection was observed against Fas-induced apoptosis in the other
cell lines (data not shown). Thus, the anti-apoptotic effect of
Toso also is limited to certain classes of hematopoietic cells,
suggesting the presence of tissue-specific mediators in these
cells.
[0144] T cell activation results in increased expression of Fas and
FasL on the cell surface. This is paradoxical, as it is clear that
T cells do not kill themselves after such induction, whereas
overexpression of Fas and FasL in other cell types does lead to
cell death. In vitro, PMA and lonomycin can induce apoptosis in T
cells (Oyalzu, et al., Biochem. Biophys. Res. Commun., 213:994-1001
(1995)) by mimicking certain aspects of CD3 engagement, including
upregulation of Fas and FasL. One function of Toso might be to
inhibit T cell activated self-killing and that the levels of Toso
might become increased following T cell activation, helping to
render Jurkat cells partially resistant to upregulated Fas and
FasL. Expression of Toso mRNA in Jurkat cells was examined by
northern hybridization. As shown in FIG. 8a, an endogenous Toso
mRNA species of 2.8 kbp was detected in resting Jurkat cells,
although expression was seen after overexposure of the blot (data
not shown). Toso mRNA expression increased, including minor species
(2.0, 3.5, 4.3, 5.5 kbp), after stimulation of Jurkat cells with
PMA and PHA (15-fold increase) or PMA in combination with lonomycin
(25-fold increase). Thus, Toso can be induced following T-cell
activation.
[0145] Jurkat.ecoR cells, Jurkat.ecoR cells infected with
pBabeMN-lacZ, and pBabeMN-Toso-infected clones were precultured
with 10 ng/ml PMA and 500 ng/ml lonomycin for 12 hours and then
incubated with 10 ng/ml of anti-Fas mAb for 24 hours, and as shown
in FIG. 8b, Jurkat cells were susceptible to anti-Fas mAb-induced
apoptosis as well as PMA/lonomycin-induced apoptosis. However,
following activation with PMA/lonomycin one third of Jurkat cells
were clearly resistant to anti-Fas mAb induced apoptosis. These
results suggest that Jurkat cells activate a protective system that
blocks Fas-mediated apoptosis, supporting the contention that
induced Toso is a mediator in this protective effect.
[0146] We further tested whether Toso expression could rescues
activation-induced programmed cell death. We randomly picked five
pBabeMN-Toso-infected Fas resistant Jurkat T cell clones and used
these to assay the inhibitory effect of Toso on
PMA/lonomycin-induced apoptosis. All five clones exhibited
significant resistance to PMA/lonomycin-induced apoptosis, as well
as continued strong resistance to Fas-induced apoptosis (FIG. 8c).
Control clones displayed the expected killing effect when activated
with PMA and lonomycin. Toso not only inhibited apoptosis activated
by Fas and TNF-.alpha., but also inhibited apoptosis induced by
certain classes of T cell activation events.
[0147] Normal T cells at early stages of activation are resistant
to Fas-induced apoptosis but become Fas sensitive at late stages of
activation (Klas, et al., (1993)). Toso expression kinetics in
peripheral blood mononuclear cells were examined after PHA
stimulation using by semi-quantitative RT-PCR. Peripheral blood
leucocyte (PBL) from healthy volunteers were isolated by
Histopaque-1077 (Sigma) density centrifugation. Adherent cells were
removed by adherence to plastic culture vessels. Cells were
activated with phytohemagglutinin (PHA)-P (1 .mu.g/ml) for 24 hours
washed, and cultured with 20 U/ml of recombinant human IL-2
(R&D Systems Inc., Minneapolis, Minn. 55413, U.S.A.). To
perform mixed lymphocyte culture, PBL were treated with 20 .mu.g/ml
of mitomycin-C (stimulating cells, SC) for 3 hours and washed. SC
were adjusted to 7.times.10.sup.5 cells/ml and cultured with an
equal volume and cell density of PBL (responding cells, RC) from
another donor (Clot, et al., Immunology, 29:445-453 (1975)). Cells
were cultured for one to seven days (day 1, 3, 5, and 7). Toso
expression was observed at day 1 and upregulated expression at day
3 after activation. However, Toso expression was clearly decreased
at days 5 (FIG. 9a), correlating with Fas sensitivity studies
(Klas, et al. (1993)). Further, allogenic stimulation in mixed
lymphocyte cultures was performed to determine whether Toso is
activated in primary immune cells upon T cell activation. As shown
in FIG. 9b, Toso expression was also rapidly induced in the
presence of stimulator cells on day 1; however Toso expression in
mixed lymphocyte cultures was reduced by day 6 to levels even lower
than seen on day 1 and responder cells alone at day 6. These
results further confirm a supportive role for Toso induced
resistance to Fas-mediated death during T lymphocyte
activation.
[0148] Natural T cell resistance to Fas-induced apoptosis shows a
time-dependent kinetics (Klas, et al. (1993)). By day 6
post-activation, T cells become susceptible to Fas-induced death.
In addition, activation of Jurkat cells by PMA/lonomycin induces a
significant increase in Fas ligand expression which is thought to
promote apoptosis (Oyalzu, et al. (1995); Brunner, et al., Nature,
373:441-444 (1995)). However, PMA/lonomycin-activated Jurkat cells
were not as efficiently induced to undergo apoptosis by anti-Fas
mAb treatment compared to unstimulated Jurkat cells (FIG. 8b). This
suggested that Jurkat cells become at least partly resistant to
anti-Fas mAb-induced apoptosis after T cell signaling, mimicking
processes observed in natural T cells. mRNA expression of Toso in
Jurkat cells, as well as in peripheral T cells, is strongly
upregulated upon stimulation with T cell activators. Further,
overexpression of Toso protected Jurkat cells against
PMA/lonomycin- induced apoptosis.
[0149] This is consistent with the proposal that Toso expression,
which transiently increased and then decreased in peripheral blood
mononuclear cells after activation with PHA or allogenic
stimulation, is responsible for the temporary Fas resistance in T
cells. Hence, the results are consistent with the hypothesis that
Toso may be involved in activation-induced resistance to apoptosis
of T cells during an immune response. We conclude from the results
that the inhibitory effect of the extracellular domain of Toso in
activation-induced apoptosis is attributable to the inhibition of
Fas-mediated signal transduction through inhibition of caspase-8 by
c-FLIP induction.
[0150] The finding that Toso can exert cell-specific and signaling
pathway specific effects on apoptosis suggests that other
polypeptides exist that act upon the Fas death induction cascade.
Critically, the fact that signalling by the extracellular domain of
Toso induces expression of cFLIP suggests the existence of a
regulatable transcription cascade that can be activated to block
Fas-mediated apoptosis in some cell types. As shown here, high
efficiency gene transduction using a retroviral approach, like
other cDNA cloning approaches (Vito, et al., Science, 271:521-525
(1996); Kitamura, et al., Prac. Natl. Acad. Sci., 92:9146-9150
(1995)), allows functional cloning of genes with high throughput
and accuracy. Further analysis of the Toso pathway coupled with
gene disruption analysis in animals will further clarify the
overall role that the extracellular domain of Toso plays in
modulating activation-induced T-cell apoptosis in vivo.
EXAMPLE 5
[0151] The Cytoplasmic Domain of Toso Promotes Cell Death in Murine
Pre-B Cells.
[0152] 70Z/3 cells were incubated with virus at 32.degree. C. for
12 hours including initial spinning and achieved 70-80% infection
efficiency estimated using FACS analysis for pBabeMN-Lyt-2. 70Z3
cells kept about 80% viability at the end of 12 hours incubation
with virus. However, after infection, we observed rapid cell death
(about 70% of cells were dead) in 70Z/3 cells infected with
pBabeMN-TOSO, not in 70Z3 cells with pBabeMN-Lyt-2 nor with
supernatant of .phi.NX-E cells (FIG. 11). Supernatant from
pBabeMN-TOSO transfected 293T cells, which is the parental cell
line of .phi.NX-E and .phi.NX-A cells, did not induce rapid cell
death to 70Z/3 cells. These results suggest that gene products of
TOSO induced rapid cell death. Most dead cells after infection
showed apoptotic nuclei under microscopic observation, suggesting
TOSO induced apoptosis to 70Z/3 cells.
[0153] To clarify which region was responsible for apoptotic signal
transduction, a set of deletion mutants of the TOSO cDNA was
prepared as shown in Example 3. The mutated TOSO cDNA was ligated
into pBabeMN retroviral vector and infected 70Z/3 cells. As shown
in Table A, below, massive cell death was observed in 70Z/3 cells
infected with pBabeMN-TOSO.HA, -TOSOA(377-390).HA, -TOSOA(334-390).
HA and Lyt-2/TOSO(271-390).HA, but not pBabeMN-TOSOA(252-390).HA
and pBabeMN-TOSOA(29-187).HA. Full length Lyt-2 did not induce
rapid cell death to 70Z/3 cells after infection (data not shown).
Lyt-2/TOSO(271-390).HA. was most effective in promoting cell death
in 70Z/3 cells, suggesting that the cytoplasmic region was
responsible for massive cell death in 70Z3 cells.
[0154] The TOSO-induced cell death in 70Z/3 cells, suggests that
TOSO works not only for protection against Fas-induced apoptosis
but also for promotion of cell death. The cytoplasmic domain from
A.sup.281 to A.sup.333 is responsible for promotion of cell death.
BLAST search reveals that this region has partial homology to FAST
kinase and acid sphingomyelinase which is involved in Fas-induced
apoptosis. When the cytoplasmic domain of TOSO is compared to the
"death domain" from several molecules, the cytoplasmic domain of
TOSO did not show any homology to known "death domain", including
the consensus sequence as described. The promotion of cell death by
TOSO was not observed in several cell lines. Cell death induced by
TOSO may be observed in some stages of B cell development.
[0155] Table A indicates the effect of TOSO deletion mutants on
promotion of apoptosis. 70Z/3 cells were infected with
pBabeMN-Lyt-2.alpha..HA, pBabeMN-TOSO.HA,
pBabeMN-TOSOA(377-390).HA, pBabeMN-TOSOA(334-390).HA,
pBabeMN-TOSOA(281-390).HA, pBabeMN-TOSOA(252-390).HA and
pBabeMN-TOSOA(29-187).HA. After infection, the stained cells were
incubated with phosphate-buffered saline including 100 .mu.g/ml of
ethidium bromide (SIGMA) and 100 .mu.g/ml of acridine orange
(SIGMA). Viable cells were identified with UV microscopy. The
percentage of viable cells is expressed as mean .+-.SD of
triplicate cultures.
2 Infected-Virus Encoding % Viable Cells Lyt-2.HA 73 .+-. 5 4.8.HA
30 .+-. 4 4.8.DELTA.(377-390).HA 31 .+-. 5 4.8.DELTA.(334-390).HA
29 .+-. 2 4.8.DELTA.(281-390).HA 75 .+-. 2 4.8.DELTA.(252-390).HA
78 .+-. 3 4.8.DELTA.(29-187).HA 83 .+-. 3 Lyt-2/4.8(271-390).HA 5
.+-. 3
* * * * *
References